Nitrogen-fixing methane-utilizing bacteria
CENTRALE LANDBOUWCATALOGUS
0000 0086 9384
Dit proefschrift met stellingenvan
JOHANNESADRIANUSMARIADEBONT,
landbouwkundig ingenieur, geboren te Waalwijk op 6maart 1947, isgoedgekeurd door depromotor,
Dr. Ir. E.G.Mulder, hoogleraar in de algemene microbiologie en de microbiologie van bodem en
water.
Derector magnificus vande Landbouwhogeschool,
Dr. Ir.J. P.H.van derWant
Wageningen,5oktober 1976
/V/V 6>Zoi
bb'J
J. A.M.de Bont
Nitrogen-fixing
methane-utilizing bacteria
Proefschrift
ter verkrijging van degraad van doctor inde
landbouwwetenschappen, op gezagvan de rector magnificus,
dr. ir. J. P.H.van derWant,hoogleraar in devirologie,
inhet openbaar te verdedigen opvrijdag 26november 1976
desnamiddagste vieruur in de aulavan de Landbouwhogeschool
te Wageningen
tSh ~ /OVOfff—C
NfJ8Zol
STELLINGEN
I
Het vermogen tot stikstofbinding is een algemeen bij mcthaanoxyderende bakterienvoorkomende eigenschap.
Dit proefschrift.
II
Acetyleen heeft een sterk remmende invloed op de oxydatie van methaan door
bakterien.
Dit proefschrift.
Ill
De acetyleen-ethyleen test ter bepaling vannitrogenase-aktiviteit kan niet toegepast
worden op bakterien die oplagere alkanen groeien.
Dit proefschrift.
IV
Vissterfte in meren tengevolge van zuurstofgebrek dat ontstaat tijdens langdurige
bedekking van het water met ijskan wellicht tegengegaan worden door geringehoeveelheden calciumcarbideonderhet ijste brengen.
J.W.M. RUDD, A.FURUTANI, R.J. FLETT and R.D. HAMILTON
(1976) Limnology and Oceanography 2 1 , 3 5 7 - 3 6 4 .
Dit proefschrift.
V
Toevoeging van acetyleen aan aardgas zalde afbraak vanmethaan in degrond random eengaslek slechtsgedurende korte tijd verhinderen.
J.A.M. de BONT (1975) Gas 95, 367-374.
VI
Tijdens de acetyleen-ethyleen test vindt er geen mikrobiologische omzetting van
ethyleen plaats.
J.A.M. DE BONT (1976) Canadian Journal of Microbiology,
22,1060-1062.
VII
Het is niet aannemelijk dat waterstof de acetyleenreduktie bij waterstofoxyderende
bakterien remt zoalsaangegeven door Gogotoven Schlegel.
J.N. GOGOTOV en H.G. SCHLEGEL (1974) Archiv fur Mikrobiologie 97, 359-362.
J.A.M. DEBONT enM.W.M.LEIJTEN (1976) Archives of Microbiology 107, 2 3 5 - 2 4 0 .
VIII
De vrijwel algemeen gedeelde meningvan Stanier, Adelberg en Ingraham dat Caulobacters in hun levenscyclus een beweeglijk stadium doormaken is in zijn algemeenheid nietjuist.
R.Y. STANIER, E.A. ADELBERG en J.L. INGRAHAM (1976)
The Microbial World. Englewood Cliffs: Prentice Hall, Inc.
J.A.M. DE BONT, J.T. STALEY en H.S. PANKRATZ (1970)
Antonie van Leeuwenhoek 36, 3 9 7 - 4 0 7 .
J.T. STALEY, J.A.M. DE BONT en K. DE JONG (1976) Antonie
van Leeuwenhoek 42, 333-342.
IX
Smith heeft zijn opvatting dat bakterien in grond slechts onder anaerobe omstandigheden ethyleen kunnen vormen onvoldoende gefundeerd.
A.M. SMITH (1976) Soil Biology and Biochemistry 8, 2 9 3 - 2 9 8 .
J.A.M. DE BONT (1975) Annals of Applied Biology 81, 1 1 9 - 1 2 1 .
J.A.M. DE BONT (1976) Antonie van Leeuwenhoek 42, 5 9 - 7 1 .
X
Abeles, Cracker, Forrence en Leather hebben zich ernstigvergist inhun berekening,
gebaseerd op hun eigen laboratoriumexperimenten, dat perjaar 7xl0 6 ton ethyleen
uit de lucht boven de Verenigde Staten van Amerika verwijderd zou kunnen worden
door mikrobiologische oxydatie van dit gasin degrond. Deuitkomst vanhun berekeningis7xl0 6 ton perjaar te hoog uitgevallen.
IV.B. ABELES, L.E. CRACKER, L.E. FORRENCE en G.R. LEATHER (1971) Science 1 7 3 , 9 1 4 - 9 1 6 .
XI
Acetylcoenzym Aiseen intermediair in de afbraak vanethyleen door bepaalde bakterien.
J.A.M. DE BONT en R.A.J.M. ALBERS (1976) Antonie van Leeuwenhoek 42, 7 3 - 8 0 .
Proefschrift vanJ.A.M.DEBONT
Nitrogen-fixing methane-utilizing bacteria
Wageningen, 26november 1976.
Promoter: Prof. Dr.Ir. E.G.Mulder
This study was carried out at the Laboratory of Microbiology, Agricultural University,
Wageningen,The Netherlands.
Dit proefschrift isvoortgekomen uit een onderzoek dat dankzij de geldelijke steun van
het VEG Gasinstituut en de N.V. Nederlandse Gasunie in het Laboratorium voorMikrobiologie van de Landbouwhogeschool uitgevoerd kon worden. Van harte dank ik devele
personen die betrokken zijn geweest bij de totstandkoming ervan. Ik ben bijzonder erkentelijk voor de begeleiding bij het in de uiteindelijke vorm brengen vandemanuskripten en voor de hulp die ik op vele andere gebieden gekregen heb. Met name wilikgraag
het tekenen van de grafieken aanhalen. Diskussies met lotgenoten, evenals de plezierige
samenwerking met doktoraalstudenten, hebikzeergewaardeerd. Evenwel,mijn dank gaat
speciaal uit naar die mensen die mijn humeur in tijden van algehele tegenspoed weer wat
opvijzelden.
VII
PREFACE
After the changeover in the Netherlands from manufactured gas to natural gas, a
strong increase in the death rate of trees in urban areas was observed. To study this
undesirable phenomenon, a committee was set up by the Netherlands Association of
Municipal Park Superintendents. This Committee for the Study of the Influence of
Natural Gas on Vegetation wasinstructed to investigate the cause of the death of treesby
natural gas and, if possible, to devise measures to prevent damage. The results of the
committee's work have been published in eight reports (SIAB reports, P.O. Box 1240,
TheHague).
For this committee, Hoeks investigated the death of vegetations brought about by
natural gas (Hoeks (1972), Thesis, Agricultural University, Wageningen; Hoeks (1972),
Soil Science 113, 46—54). He found that leakage of naturalgasfrom the gas distribution
system affects the physical, chemical and biological processesinthe soil,the most important of these processes being the microbial oxidation of methane. In this oxidation process oxygen was consumed and carbon dioxide wasproduced.When the temperature was
not too low,the rate of oxygen consumption in soilaround gasleakswassointensive that
the supplied oxygen was fully consumed. Absence of oxygen from the soil atmosphere
wasseen asthe main cause of damage to the vegetation.
Thus it was decided to examine more closely the organisms involved in the microbial
oxidation of natural gas. A program of research was initiated by Prof. Mulder and Dr
Adamse, both advisory members of the Committee for the Study of the Influence of
Natural Gas on Vegetation. As aresult,apublication on the microbiology of processesin
soil near gas leaks appeared in 1972 (Adamse, Hoeks, de Bont and van Kessel (1972),
Archiv fur Mikrobiologie 83,32—51).Methane-oxidizing bacteria were found to be mainly responsible for the increased oxygen consumption insoil around leaks.
Since then, attention has been focussed on the methane-oxidizing bacteria. It was
observed that these bacteria have the capacity to utilize atmospheric nitrogen gas for
synthesis of cell material. Nitrogen fixation by these organismswasthought to be important in explaining some of the processes occurring in soil around a gas leak (de Bont
(1975),Gas95,367—374).Consequently,thisimportantpropertywasinvestigated in more
detail. The results obtained on nitrogen-fixation by methane-utilizing bacteria are presented in thisthesis.
IX
CONTENTS
Preface
IX
General introduction
1
Paper I
Nitrogen fixation and co-oxidation of ethylene by amethane-utilizing bacterium
Journal of General Microbiology (1974),83, 113-121
7
Paper II
'
Invalidity of the acetylene reduction assay in alkane-utilizing, nitrogen-fixing
bacteria
Applied and Environmental Microbiology (1976),31, 640—647
17
PaperIII
Nitrogen fixation by methane-utilizing bacteria
Antonie van Leeuwenhoek (1976),42, 245-254
31
Paper IV
Hydrogenase activity in nitrogen-fixing methane-oxidizing bacteria
Antonie van Leeuwenhoek (1976),42,255—260
41
General discussion
45
Summary
55
Samenvatting (Summary inDutch)
59
XI
GENERAL INTRODUCTION
In nature, methane is continuously produced by bacteria as an end product of anaerobic mineralization of organic matter. Methane-producing bacteria may be active in
sediments of lakes, ditches and rivers,submerged soils,anaerobic digestersused insewage
purification and also in ruminant digestion. Methane also occursin coal and oil deposits;
naturalgascontains mainly methane.
In view of the natural occurrence of the gas it is not surprising that bacteria have
developed the ability to utilize methane as carbon and energy source. Sohngen (1906)
demonstrated for the first time that methane is susceptible to bacterial action. From
experiments with water plants he concluded that bacteria associated with the plants
rather than the plantsthemselveswere responsible for the uptake of methane.Heisolated
Bacillusmethanica, a Gram-negative, short, thick rod as wellasother bacteria capable of
oxidizing methane. Fifty years elapsed after the isolation and description of these
methane-oxidizing bacteria by Sohngen, before Foster and co-workers reported their
studies on methane oxidation (Dworkin and Foster, 1956; Leadbetter and Foster, 1958;
Leadbetter and Foster, 1960; Foster and Davis, 1966). Bacillus methanica, originally
isolated by Sohngen, was reisolated along with other strains resembling this bacterium.
Also, a coccus capable of oxidizing methane wasisolated. These workers found that only
methane and methanol served as growth substrates for their isolates. The inability of
methane-oxidizing bacteria to grow on a variety of substrates other than methane or
methanol has since been confirmed in many studies (Stocks and McClesky, 1964;Brown,
Strawinski and McClesky, 1964; Whittenbury, Phillips and Wilkinson, 1970;Hazeu and
Steennis, 1970; Adamse, Hoeks, de Bont and vanKessel, 1972;Namsaraev and Zavarzin,
1972; Hazeu, 1975). From these studies it also became evident that morphologically the
methane-oxidizing bacteria form a heterogeneous group. Non-motile cocci, motile rods,
vibrios and pear-shaped organisms and pleomorphic strains were isolated. Often these
bacteria were capable of existing in forms resistant to adverse environmental conditions.
Exospores and different types of cysts have been observed (Whittenbury, Davies and
Davey, 1970). Although the methane-oxidizing bacteria show much variation in morphology, their internal anatomy is more uniform. The presence of a system of internal
membranes in these bacteria has been described by Procter, Norris and Ribbons (1969)
and later by others (Davies and Whittenbury, 1970; Smith and Ribbons, 1970; Smith,
Ribbons and Smith, 1970; de Boer and Hazeu, 1972). These membranes may either be
distributed throughout the cytoplasm of the cell (type I) or peripheral (type II). The
type I membranes have been found in the rod and coccoid organisms and type II membranesin the vibrioid organisms.
The occurrence of these intracytoplasmatic membrane systems in methane-oxidizing
bacteria suggeststhat they support physiological andbiochemical activities unique for the
growth of these bacteria. These membranes may beinvolved inthe oxidation of methane
to the level of formate. The first step in the oxidation of methane isprobably catalysed
by a monooxygenase, yielding methanol as the reaction product. This oxidation activity
dependent on NADH and 0 2 (Ribbons and Michelover, 1970; Ferenci, 1974) is associated with particulate cell-free fractions that contains membranous material (Ribbons,
1975). Further oxidation of methanol through formaldehyde to formate has also been
associated with the internal membranes (Wadzinkski and Ribbons, 1975),although soluble dehydrogenase activity for these two substrates has been demonstrated aswell(Patel
and Hoare, 1971;Patel, Bose, Mandy and Hoare, 1972). The final step in the oxidation
of methane to carbon dioxide iscatalysed by asoluble formate dehydrogenase (Patel and
Hoare, 1971;Wadzinkski and Ribbons, 1975).
The synthesis of cell constituents by methane-oxidizing bacteria necessarily involves
the condensation of Ci-carbon units derived from methane. This requires special biosynthetic capabilities enabling the formation of C3 units. Once a C3 unit has been
formed, the more complex molecules may be synthesized alongmetabolic routes that are
identical to those encountered in other bacteria. Two completely different pathways for
the formation of C3 units from Ci units have been found in methane-utilizing bacteria.
The ribose phosphate cycle of formaldehyde fixation wasfirst discovered inPseudomonas
methanica. The first enzyme operative in this carbon-assimilating pathway is hexulosephosphate synthetase catalysing the formation of D-arabinose-3-hexulose-6-phosphate
from ribulose-5-phosphate and formaldehyde originating from the methane molecule
(Kemp, 1974). A cycle of reactions then regenerates the ribulose-5-phosphate and yields
C3 compounds in amounts equivalent to the amount of formaldehyde that has been
fixed (Quayle, 1972). Alternatively, via the serine pathway both formaldehyde and
carbon dioxide originating from methane may be incorporated into a C3 compound.
Serine transhydroxymethylase catalyses the condensation of formaldehyde with glycine
yielding serine, while phosphoenol pyruvate carboxylase catalyses the incorporation of
carbon dioxide into phosphoenol pyruvate giving rise to oxaloacetate. Glycine and oxaloacetate are regenerated in a cycle of reactions and the net result is the formation of
3-phosphoglycerate from 2molecules of formaldehyde and 1molecule of carbon dioxide
(Quayle, 1972).
A provisional classification of the methane-oxidizing bacteria into groups and subgroups, based on morphology, fine structure and type of resting stage formed, has been
Table 1. Division of methane-oxidizing bacteria into groups according to Whittenbury,
Phillips and Wilkinson (1970).
Group
Methylosinus
Methylocystis
Methylomonas
Methylobacter
Methylococcus
Resting stage
Membrane type Morphology
Exospore
Lipid cyst
Immature Azotobacter-type cyst
Azotobacter-type cyst
Immature Azotobacter-type cyst
II
II
I
I
I
Rod or pear-shaped
Rod or vibrio
Rod
Rod
Coccus
proposed by Whittenbury,Phillipsand Wilkinson (1970). Table 1showsthis classification
into groups. Later Lawrence and Quayle (1971) found that a correlation exists between
the membrane type of the bacteria and the assimilatory pathway. Organisms possessinga
type I membrane structure use the ribose phosphate cycle of formaldehyde fixation,
whereas those possessing atype IImembrane structure use the serine pathway.
Nitrogen fixation
A wide range of procaryotic microorganisms, including many types of heterotrophic
and photoautotrophic bacteria and species of blue-green algae, have the ability to fix
atmospheric nitrogen (Quispel, 1974). The N2 fixation in all these organisms is accomplished by the catalytic action of the nitrogenase enzyme system. This enzyme system
consists of a protein containing Fe and aprotein containing Feand Mo.Cell-free preparations capable of fixing N2 have been prepared from a number of organisms. From such
preparations it has been established that the nitrogenase reaction iscatalysed by acombination of the two proteins in which ATPhydrolysis iscoupled to electron transfer for the
reduction of N2 to NH 3 . Nitrogenase reducesmany substrates other than N 2 . Reduction
of acetylene to ethylene isparticularly important because aconvenient and sensitive assay
system for nitrogenase activity has been based on this reaction. This acetylene-ethylene
assay (Hardy, Burns and Holsten, 1973) can be employed for studying activity of both
enzyme preparations and intact organisms. It enables not only a ready estimation of the
nitrogen-fixing capacity of pure cultures under laboratory conditions, but it can also be
used for measuring nitrogen-fixing activities in natural habitats.
Free-living bacteria capable of fixing nitrogen are mainly found amongst the obligate
and facultative anaerobes. Obligate aerobic nitrogen-fixing bacteria are much less frequently encountered, presumably because of the reductive character of the nitrogen-fixing process. The family Azotobacteraceae is the best known group of obligate aerobic
free-living nitrogen-fixing bacteria (Mulder and Brotonegoro, 1974). Bacteria of the type
of Mycobacterium flavum (Federov and Kalininskaya, 1961;de Bont and Leyten, 1976)
form asecond group of aerobic nitrogen fixers.
There is much confusion in the literature about the ability of the obligate aerobic
methane-oxidizing bacteria to fix atmospheric nitrogen. Indirect evidence for nitrogen
fixation by these bacteria has been available ever since Schollenberger (1930) and Harper
(1939) made their observations on the effect of natural gason soil.Theynoticed that soil
exposed for a prolonged period of time to natural gasescaping from leaking pipe lineshad
a considerably higher nitrogen content than unexposed control soil.Comparable observations were made by Hoeks (1972) in a study on the effect of natural gas leaking from
mains on the composition of soil air. Since methane is the main component of natural
gas, and since this hydrocarbon had a similar effect on the nitrogen content of soil
(Davis, Coty and Stanley, 1964; Coty, 1967), methane-oxidizing bacteria capable of
fixing nitrogen were thought to be responsible. Coty alsoperformed an experiment with
15
N2 using an isolated culture instead of soil, but the purity of this culture may be
questioned as it also grew on nutrient agar. Employing the acetylene-ethylene assay,
Whittenbury, Phillips and Wilkinson (1970) obtained inconclusive results while Adamse,
Hoeks, de Bont and van Kessel (1972) concluded that their strain did not fix elementary
nitrogen, although growth wasobtained inanitrogen-free medium.
The purpose of the work for this thesiswasto obtain more information about possible
nitrogen fixation by the methane-oxidizing bacteria.
Outline of the investigations
In this thesis the investigations onnitrogen fixation by methane-oxidizing bacteria are
reported in four papers.
Paper I deals with the ability of a pure culture of the methane-oxidizing bacterium
strain 41 to fix atmospheric nitrogen. With 1 5 N 2 , it was demonstrated that thisMethylosinus type bacterium converted the gas into cell material. However, nitrogenase activity could not be assayed by acetylene reduction when the bacterium was growing on
methane. The organism was sensitive to oxygen when dependent on N2 as nitrogen
source.
Paper II comprises a study of the erratic behaviour of the acetylene-ethylene assay
with strain 41. This study included other strains of methane-oxidizing bacteria and
bacteria growing on higher alkanes.
Paper HI givesthe results of asurvey of the nitrogen-fixing capacity amongst methaneoxidizing bacteria. This study could be undertaken with the results obtained in paperI
(oxygen sensitivity) and paper II (erraticbehaviour of the acetylene-ethylene assay).
Paper IV draws attention to hydrogenase activity in strain 41. This activity wasassociated with nitrogenase activity.
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NITROGEN FIXATION ANDCO-OXIDATION OFETHYLENEBY A
METHANE-UTILIZINGBACTERIUM
By J. A.M.DEBONT and E.G.MULDER
Journal of General Microbiology 83 (1974) 1 1 3 - 1 2 1
A methane-oxidizing bacterium, isolated from soil, was capable of fixing
nitrogen. Nitrogenase activity could be assayed by acetylene reduction when
the bacterium was growing on methanol but not when growing on methane,
although 1SN2 was fixed. Bacteria growing on methane co-oxidized ethylene
but methanol-growing cells did not. The organism was extremely sensitive to
oxygen when dependent on N2 as nitrogen source, a consequence of the
sensitivity of itsnitrogenase towards oxygen.
INTRODUCTION
Nitrogen accumulates in soil exposed to natural gas (Schollenberger, 1930; Harper,
1939). Since methane is the main component of natural gas, and sincethis hydrocarbon
had a similar effect on the nitrogen content of the soil (Davis, Coty &Stanley, 1964;
Coty, 1967), methane-oxidizing bacteria, capable of fixing nitrogen, were thought to be
responsible. Methane-oxidizing bacteria capable of growing in nitrogen-free medium were
isolated. Nitrogen fixation was measured by Kjeldahl analyses of cultures incubated for
periods of up to four months (Davis et al. 1964), whilst Coty (1967) performed an
experiment with 1S N 2 . As the isolated cultures also grew on nutrient agar,the purity of
the cultures may be questioned. Whittenbury, Phillips & Wilkinson (1970) isolated a
straiit of Methylosinus trichosporium from Coty's culture,whichwasable to reduce25%
of the acetylene present during an incubation period of 7 to 14days. All of the other
strains of methane-oxidizing bacteria tested reduced very little acetylene.Wereport here
atmosphericnitrogen fixation by arecently isolated methane-oxidizing bacterium.
METHODS
Medium. The mineral-salts solution (MSmedium), usedthroughout the investigation,
contained the following salts in 11 deionized water: NaN0 3 , 2-0g; K 2 HP0 4 , 0-5g;
KH 2 P0 4 , 0-5g; MgS0 4 -7H 2 0, 0-2g; CaCl2, 0-015 g; FeS0 4 -7H 2 0, 0-001g; CuS0 4 5H 2 0, 5jug; H3BO3, lOjitg; ZnS0 4 -7H 2 0, 70/ug; MnS0 4 -5H 2 0, 10/ig; Na 2 Mo0 4 -
DAVIS,J. B., COTY, V. F. and STANLEY, J. P. (1964). Atmospheric nitrogen fixation by methaneoxidizing bacteria. Journal of Bacteriology 88, 4 6 8 - 4 7 2 .
DROZD, J. and POSTGATE, J. R. (1970). Interference by oxygen in the acetylene-reduction test for
aerobic nitrogen-fixing bacteria. Journal of General Microbiology 60, 4 2 7 - 4 2 9 .
FAUST, H. (1967). Probenchemie 1 5 N markierter Stickstoffverbindungen im Mikro- bis Nanomolbereich fur die emissionsspektrometrische Isotopenanalyse. Isotopenpraxis 3, 1 0 0 - 1 0 3 .
FERRARIS, M. M. and PROKSCH, G. (1972). Calibration methods and instrumentation for optical
,5
N determinations with electrodeless discharge tubes. Analytica chimica acta 59, 177-185.
HARDY, R. W. F., BURNS, R. C. and HOLSTEN, R. D. (1973). Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biology and Biochemistry 5, 4 7 - 8 1 .
HARPER, H.J. (1939). The effect of natural gas on the growth of micro-organisms and the accumulation of nitrogen and organic matter in the soil. Soil Science 48, 4 6 1 - 4 6 6 .
HAZEU, W. and STEENNIS, P. J. (1970). Isolation and characterization of two vibrio-shaped niethane-oxidizing bacteria. Antonie van Leeuwenhoek 36, 6 7 - 7 2 .
HERBERT, D., PHIPPS, P. J. and STRANGE, R. E. (1971). Chemical analysis of microbial cells. In
Methods in Microbiology, vol. 5B, pp. 209—344. London and New York: Academic Press.
HILL, S. (1971). Influence of oxygen concentrations on the colony type of Derxia gummosa grown
on nitrogen-free media. Journal of General Microbiology 6 7 , 7 7 - 8 3 .
.
LARGE, P.J. and QUAYLE, J.R. (1962). Microbial growth on CI compounds. V. Enzyme activities in
extracts of Pseudomonas AMI. Biochemical Journal 87, 386-396.
LAWRENCE, A. J. and QUAYLE, J. R. (1970). Alternative carbon assimilation pathways in metihaneutilizing bacteria. Journal of General Microbiology 63, 371-374.
NAMSARAEV, B. B. and ZAVARZIN, T. A. (1972). Trophic relationships in a methane-oxidizing
culture. In Mikrobiologiya 4 1 , pp. 999-1006, English translation pp. 887-892. New York:
Plenum Publishing Corp.
PAREJKO, R. A. and WILSON, P. W. (1971). Kinetic studies on Klebsiella pneumoniae nitrogenase.
Proceedings of the National Academy of Sciences of the United States of America 68, 2016-2018.
POSTGATE, R. (1971). Biochemical and physiological studies with free-living, nitrogen-fixing bacteria. Plant and Soil, Special Volume 1971, 5 5 1 - 5 5 9 .
QUAYLE, J. R. (1972). The metabolism of one-carbon compounds by microorganisms. Advances in
Microbial Physiology 7, 119-203.
SCHOLLENBERGER, C. J. (1930). Effect of leaking natural gas upon the soil. Soil Science 29,
261-266.
WHITTENBURY, R., PHILLIPS, K. C. and WILKINSON, J. F. (1970). Enrichment, isolation and
some properties of methane-utilizing bacteria.Journal of General Microbiology 61, 205-218.
NITROGEN FIXATION AND CO-OXIDATION OF ETHYLENE BY A
METHANE-UTILIZING BACTERIUM
ByJ. A.M.DE BONT and E.G.MULDER
Journal of GeneralMicrobiology 83(1974) 113-121
A methane-oxidizing bacterium, isolated from soil, was capable of fixing
nitrogen. Nitrogenase activity could be assayed by acetylene reduction when
the bacterium was growing on methanol but not when growing on methane,
although 15 N 2 was fixed. Bacteria growing on methane co-oxidized ethylene
but methanol-growing cells did not. The organism was extremely sensitive to
oxygen when dependent on N2 as nitrogen source, a consequence of the
sensitivity of its nitrogenase towards oxygen.
INTRODUCTION
Nitrogen accumulates in soil exposed to natural gas (Schollenberger, 1930; Harper,
1939). Since methane is the main component of natural gas, and sincethis hydrocarbon
had a similar effect on the nitrogen content of the soil (Davis, Coty &Stanley, 1964;
Coty, 1967), methane-oxidizing bacteria, capable of fixing nitrogen, were thought to be
responsible. Methane-oxidizing bacteria capable of growingin nitrogen-free medium were
isolated. Nitrogen fixation was measured by Kjeldahl analyses of cultures incubated for
periods of up to four months (Davis et al. 1964), whilst Coty (1967) performed an
experiment with 15 N 2 . As the isolated cultures also grew on nutrient agar,the purity of
the cultures may be questioned. Whittenbury, Phillips & Wilkinson (1970) isolated a
strain of Methyiosinus trichosporium from Coty's culture,which wasable to reduce25%
of the acetylene present during an incubation period of 7 to 14days. All of the other
strains of methane-oxidizing bacteria tested reduced very little acetylene.Wereport here
atmospheric nitrogen fixation by arecently isolated methane-oxidizing bacterium.
METHODS
Medium. The mineral-salts solution (MSmedium), usedthroughout the investigation,
contained the following salts in 11 deionized water: NaN0 3 , 2-0g; K 2 HP0 4 , 0-5g;
KH 2 P0 4 , 0-5g; MgS0 4 7H 2 0, 0 2 g; CaCl2, 0015 g; FeS0 4 -7H 2 0, 0-001g; CuS0 4 5H 2 0, 5jug; H3BO3, 10/ig; ZnS0 4 -7H 2 0, 70jug; MnS0 4 -5H 2 0, lO^g; Na 2 Mo0 4 -
2H 2 0, 100/ng; final pH, 6-8. For solid media 1-5% agar was added to this nutrient
solution.
Chemicals. Methane, ultra pure (99-97%), and CH3OCH3, said to be 99-87% pure
(see later), were obtained from the Matheson Co., and lithium hydroxypyruvate, 98%
pure,from Sigma.
Isolation andgrowth of the bacterium. Garden soilwasincubated at 28 ±2°C under
10%methane in air. After 25 days, serial dilutions were made in MSmedium, and after
another 4weeks under 10%methane,material of the highest dilution showinggrowthwas
streaked on plates of MSagar. Well-developed colonies had grown on these plates after
3 weeks of incubation under 10%methane. Pure cultures, obtained after restreaking on
the same medium, were then tested for methane-oxidizing capacity by growing the bacterium on slants of MSwith and without methane. Weekly subcultures on slants of MS
and slants of MS without nitrate were maintained in a desiccator in which the oxygen
tension was lowered by flushing withnitrogen.Methane wasinjected through aSuba-seal
mounted in a rubber stopper. Growth rate wasmeasured in stirred 350mlside-arm flasks
sealed with rubber stoppers,fitted with Suba-seal caps.Celldensity wasmeasured with an
EEL nephelometer. During assays of acetylene reduction the cultures were stirred on a
New Brunswick Scientific G 10gyratory shaker at 150 rev./min.
Preparationof cell-freeextract. Washed cells,suspended in0-03M-sodium phosphate
buffer, were disrupted by ultrasonic disintegration. The extract was centrifuged and the
supernatant fluid used for assay.
Enzyme assays. Hydroxypyruvate reductase (EC. 1.1.1.29) and glyoxylate reductase
(EC. 1.1.1.26) were assayed in cuvettes (3ml, light path 1cm) containing 100/xmol
phosphate buffer, pH 6-8,0-4/xmolNADHand extract in atotal volume of 3ml. Lithium
hydroxypyruvate (2iimol) or sodium glyoxylate (2jumoi)were added and the decreasein
extinction at 340 nmwasmeasured against ablank containing buffer, NADHand extract.
Determination of protein. The Folin-Ciocalteu method was used (Herbert, Phipps&
Strange, 1971).
Gas-chromatographic analyses. Analyses were carried out at 50°C with a Becker
Multigraph type 407 gas chromatograph. Oxygen and nitrogen were measured by thermal
conductivity using a 200 cm x 4mm column containing a 13X molecular sieve
(60—80mesh). Hydrogen was used as carrier gas.Methane,dimethylether, and reduction
of acetylene were assayed with a flame-ionization detector, using a 110cm x 4mm
Porapak R column with nitrogen ascarriergas.
1S
N analyses. Sample preparation was a modification (Akkermans, 1971) of the
method of Faust (1967). Optical N-analyses were made with a 15N analyser (Statron
NOI-4). The method of Ferraris & Proksch (1972) was used for calculating the 15 N
content.
RESULTS
Descriptionof the bacterium. Theisolate used, strain 41, isacurved rod,motile in young
cultures (Fig. 1). On ageing of the culture, motility is lost and exospores are formed. In
old cultures, only exospores and lysed cells were found (Fig.2). Morphologically, the
organism differs slightly from the isolates of Adamse, Hoeks, de Bont & van Kessel
(1971) and of Namsareav&Zavarzin (1972),but resembles one of the types described by
Whittenbury et al. (1970) (Methylosinus sporium), and by Hazeu & Steennis (1970)
(Methylovibrio sohngenii). Purity of the strain was confirmed by microscopic examination and by streaking the culture onto various media for heterotrophs without obtaining
growth. On plates of MS, uniform colony development was seen under methane. On
slants of MS, without methane added, growth wasjust visible macroscopically and the
bacteria present resembled strain 41 morphologically. A second transfer did not grow.
Attempts to promote growth without methane by the addition of small quantities of
yeast extract or peptone were unsuccessful. Higher concentrations of these and other
Fig- 1
Fig. 2
Fig. 1. Strain41from afive-dayculture,grownonslantsofMSunder 10%CH„.
Fig.2. Strain 41, exospores and lysed cells, from athree-week culture,grownon slants
of MS under 10%CH„.
25
25
(a)
(b)
20
20
315
15
Ir
10
10
^
I
2
Time(h)
I
2
Time(h)
Fig. 3. Effect of strain 41 culture, growing in nitrogen-free MS with 10% CH4 (a) and
0-1% CH 3 OH (b), on ethylene (13 p.p.m.) in the absence (•) and presence (o) of 10%
C 2 H 2 . Ethylene concentrations were measured in 50 ml flasks with 10 ml growing
cultures. The gas phase in the flasks consisted of 5% 02, 10%CH 4 and 85%N 2 (a) and
5% O, and 95%N, (b) before the introduction of ethylene and acetylene.
substrates completely prevented growth of the first transfer. Strain 41 also utilized
CH3OH for its development. No growth occurred with C 2 H 2 , C2H4, C 2 H 6 , CH3OCH3,
C2H5OH, glucose or acetate when added as potential substrates.No definite conclusions
can be drawn for CH3OCH3. Our gas-chromatographic analysis of the contents of the
cylinder containing CH3OCH3 (99-87% pure according to the supplier) indicated the
presence of about 5%methane and approximately 0-5% of an unidentified compound
among other minor impurities. Utilization of CH3OCH3 by methane-oxidizing bacteria
has been reported and itsrole inthe biologicalbreakdown of methane discussed (Quayle,
1972).
Hydroxypyruvate reductase, the key enzyme of the serine pathway (Large &Quayle,
1962), was present in strain 41.The activities were 21jumol NADH oxidized/h/mg protein for hydroxypyruvate and 1-5for glyoxylate.
Growth in nitrogen-freemedium. If incubated without shaking, strain 41 grew slowly in
MS medium under methane when nitrate was omitted from the medium. Acetylenereduction assays for nitrogenase activity gave negative results. Growth in nitrogen-free
10
1
2
1
2
Time (days)
Time (days)
Fig.4. Growth of strain 41 on CH4 as influenced by 0 2 . Portions (10 ml)of aculture
growing logarithmically were injected into 350ml side-arm flasks containing 10% CH4
and varying concentrations of 0 2 and N2. 0 2 consumption during the experiment was
less than 2%. (a) Growth in nitrate-free medium: •, 4%0 2 ; o, 6%0 2 ; x, 8%0 2 ; •,11%
0 2 . (b)Growth withnitrate: •, 6%0 2 ;o, l l % 0 2 ; x , 15%0 2 .
medium was strongly promoted by lowering the oxygen tension, although no acetylene
reduction was detected. When growing the organism in the same medium with methanol
as substrate, ethylene production wasclearly demonstrated after 10%acetylene hadbeen
introduced. The amount of ethylene formed could quantitatively account for the growth
utilizing nitrogen fixation. The apparent difference in acetylene reduction may be due to
the ability of CH4-grown cellsto remove ethylene from the gasphase.After introduction
of acetylene into such a gasphase,the ethylene concentration remained at the samelevel
(Fig. 3a).Cells grown on methanol did not affect ethylene (Fig. 3b).
Effect of oxygen. Growth of strain 41 in liquid MS medium with methane was little
influenced by oxygen tensions of between 4 and 20%. On nitrogen-free medium, however, increasing the oxygen tension severely reduced growth (Fig.4a,b). Similar phenomena were observed when the bacterium was grown on agar plates of MSunder methane.
Colony size after three weeks of incubation was not influenced by varying the oxygen
tension. But when nitrogen-free MS was used, the effect of oxygen became apparent.
Under 20%oxygen only meagre growth developed. Growth wasbetter in restricted areas
of such a plate (Fig. 5). Incubation in 5% oxygen allowed normal development on
nitrogen-free medium (Fig.6). That the sensitvity towards oxygen is directly related to
the nitrogenase activity was shown by measuring acetylene reduction by methanol-grown
11
Fig. 5
Fig. 6
Fig. 5. Growth of strain 41 on nitrogen-free MS agar medium under 20% 0 2 , 10% CH4
and 70%N2 after 3weeks of incubation, showing sparse growth except at the edgesof
thestreaks.
Fig.6. Growth of strain 41 on nitrogen-free MSagar medium under 5%0 2 , 10% CH„
and 85% N2 after 3weeksof incubation.
cells exposed to varying oxygen tensions (Fig. 7). In a subsequent experiment, nitrogenase was inactivated by exposure to 28%oxygen in the atmosphere and activity restored
after lowering the oxygen tension (Fig.8).
The behaviour of strain 41 towards oxygen (Fig.4a, b) resembles that of certain
Azotobacter species (Dalton &Postgate, 1969).Both types of bacteria are highly sensitive
to moderate concentrations of oxygen when depending on N2 as nitrogen source, but
insensitive to oxygen inthe presence of combined nitrogen.The poor growth of strain 41
under methane on a nitrogen-free agar medium when exposed to air, in contrast to
normal growth at decreased oxygen tension (Figs.5 and 6), agrees with a similar behaviour of the nitrogen-fixing Derxiagummosa (Hill, 1971).In both organismsthe nitrogenase system does not function when the agar plates are kept in air, but normal nitrogen
fixation occurs at reduced oxygen pressure. Sporadically occurring nitrogen-fixing colonies may arise on plates exposed to air as a result of locally occurring clumps of the
inoculated organism. Oxygen supply to cells in the interior of the clumps would be
lowered, so the development of the nitrogenase systemwould become possible. Nitrogenfixing colonies are often seen at the end of an inoculation streak of anitrogen fixer where
accumulation of cellseasilyoccurs.
A response similar to that of nitrogenase of strain 41 towards oxygen (Figs.7 and 8)
has been found for nitrogenase of Azotobacter chroococcum (Postgate, 1971;Drozd &
Postgate, 1970).
12
Table I. 1SJV2 fixation by strain 41
Blank
Sample 1
Sample 2
Atom%lsN
Time of
incubation (h)
Nephelometer
readings
Suspension
(ml)
Nitrogen/
sample
(ng)
Measured
Theoretical*
0
8
23
9
13
24
70
40
30
180
134
225
0-37
1-84
5-13
3-45
6-62
_
* See results.
80
100
60
75
6,
|
£40
50
/
25 -
20-
50
100
Time (min)
Fig. 7
150
./
1
2
Time (h)
Fig. 8
Fig. 7. Acetylene reduction as influenced by 0 2 . Portions (10 ml) of a culture growing
under reduced 0 2 tension on CH 3 OH in nitrogen-free MSwere injected into 50 ml flasks
containing 10% C 2 H 2 and different levels of 0 2 and N 2 . •, 4-4% 0 2 ; o 6-3% 0 2 ; x,
10-2% 0 2 ; • , 18-0% 0 2 .
Fig. 8. Reactivation of nitrogenase, inactivated after exposure to high 0 2 concentration. Thirty ml of a culture growing under reduced 0 2 tension on CH2OH in nitrogenfree MS was injected into a 100 ml flask containing 10% C2H 2 , 4-5% 0 2 and 85-5% N 2 ,
and ethylene production measured for 1h. Excess 0 2 was then injected. After releasing
pressure, the 0 2 concentration was 28%. After 45 min the flask was opened, flushed
with N 2 , and C2H2 was restored to 10%;0 2 tension was now 3-5%.
13
Nitrogen fixation on methane. Evidence for nitrogen fixation by strain 41when growing
with methane was obtained with 15 N 2 (Table 1). The bacteria were grown in nitrogenfree medium at reduced oxygen tension. In early logarithmic phase, 70mlof the culture
was injected into a 250 ml flask. Another 70 mlwasused asablank. Thegasphase in the
flask consisted of 20% methane, 5% oxygen and 75% nitrogen. The nitrogen gas was
enriched with 10% 15 N. After 8h,40 ml of the sample waswithdrawn and the gasphase
inside the flask was brought back to atmospheric pressure with helium. The second
sample wastaken after the culture had grown for 23 h.
After correcting for the blank, the excess 15N percentages of 1-47 and 4-76 in the
culture were of the same comparative magnitude asvaluesof 3*08and 6*25, respectively,
calculated by using nephelometer readings as a measure for nitrogen assimilation in
growing cultures.The lowvalue for the first sample could be due to dissolved nitrogengas
in the culture when it wasplaced under the gasphase enriched with 15 N 2 .
Additional evidence for nitrogen fixation was obtained by growing strain 41 in
nitrogen-free medium under, initially, 10%methane and 6%oxygen, with helium/nitrogen gas mixtures to give different nitrogen pressures. Fig.9 shows that under these
conditions, growth increased as the N2 increased. From this experiment, it was estimated
that the Km value for N2 willbe approximately 16%.Thisvalue isin agreement with Km
constants found for crude extracts of other nitrogen-fixing bacteria (Parejko &Wilson,
1971).
0
2
Time (days)
Fig.9. Effect of N2 on the growth of strain 41 in nitiogen-free MS medium with
methane. Portions (10 ml) of a culture growing in nitrogen-free MS under CH4 were
injected into 350 ml side-arm flasks with 6%0 2 , 10%CH4 and different concentrations
of N, and Heto 1 atm. •, 5-7% N 2 ; o, 11-9%N,; x, 20-4%N 2 ; •, 80-6%N,.
14
1
DISCUSSION
Strain 41 has the physiological characteristics of amethane-oxidizing bacterium. Only
methane and methanol support growth. The presence of hydroxypyruvate reductase indicated that synthesis of cellular material proceeded by the serine pathway (Lawrence&
Quayle, 1970).
Measurement of nitrogen fixation by methane-oxidizing bacteria during prolonged
periods of incubation (Davis et at 1964) is open to criticism. Uptake of 1SN2 by one of
the isolated strains (Coty, 1967) provided more convincing evidence of the nitrogen
fixation. The fact that the isolated culture grew on nutrient agarsuggeststhat itwasnot
pure. This was corroborated by the isolation of a strain ofMethylosinus trichosporum
from Coty's culture (Whittenbury et al. 1970). Upon exposure to an atmosphere containing 4-4% methane and 1-8%acetylene for 7 or 14days, this isolate had reduced the
acetylene concentration by 25%.It is unknown whether the disappearance of acetylene
was due to co-oxidation of acetylene or to reduction of this compound to ethylene;data
on ethylene production were not reported. From this result no definite conclusions can
be drawn concerning the nitrogen-fixing ability of the organism,because only short-term
exposure times should be used in the acetylene-reduction technique for assessing presence
of nitrogenase (Hardy, Burns&Holsten, 1973).
Our results demonstrate fixation of atmospheric nitrogen by the methane-oxidizing
strain 41.Nitrogenase activity, measured by acetylene reduction, could only be detected
when the bacteria were growing on methanol without combined nitrogen. Co-oxidation
of ethylene by cultures grown under methane and acetylene may explain the apparent
lack of ethylene production from acetylene. Alternatively, acetylene may block methane
oxidation and thus prevent the supply of ATPto nitrogenase.
The authors are much indebted to Dr A.D.Adamse for his continuous interest and
criticism during the course of the investigation and in the preparation of the manuscript.
We thank Dr A.D.L. Akkermans for his stimulating discussions and for performance of
the 15N analyses. Support for the research has come in part from grants of the N.V.
Nederlandse Gasunie and the V.E.G. Gasinstituut.
REFERENCES
ADAMSE, A. D., HOEKS,J., DE BONT, J. A. M. and VAN KESSEL, J. F. (1971). Microbial activities
in soil near natural gas leaks.Archiv fur Mikrobiologie 83, 3 2 - 5 1 .
AKKERMANS, A. D. L. (1971). Nitrogen fixation and nodulation of Alnus and Hippophae under
natural conditions. Thesis, University of Leiden, The Netherlands.
COTY, V. F. (1967). Atmospheric nitrogen fixation byhydrocarbon-oxidizingbacteria. Biotechnology
and Bioengineering 9, 2 5 - 3 2 .
DALTON, H. and POSTGATE, J. R. (1969). Effect of oxygen on growth of Azotobacter chroococcum in batch and continuous cultures.Journal of General Microbiology 54, 4 6 3 - 4 7 3 .
15
DAVIS,J. B., COTY, V. F. and STANLEY, J. P. (1964). Atmospheric nitrogen fixation by methaneoxidizing bacteria.Journal of Bacteriology 88, 468-472.
DROZD, J. and POSTGATE, J. R. (1970). Interference by oxygen in the acetylene-reduction test for
aerobic nitrogen-fixing bacteria. Journal of General Microbiology 60, 4 2 7 - 4 2 9 .
FAUST, H. (1967). Probenchemie 1 5 N markierter Stickstoffverbindungen im Mikro- bis Nanomolbereich fur die emissionsspektrometrische Isotopenanalyse. Isotopenpraxis 3, 1 0 0 - 1 0 3 .
FERRARIS, M. M. and PROKSCH, G. (1972). Calibration methods and instrumentation for optical
15
N determinations with electrodeless discharge tubes. Analytica chimica acta 59, 177-185.
HARDY, R. W. F., BURNS, R. C. and HOLSTEN, R. D. (1973). Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biology and Biochemistry 5, 4 7 - 8 1 .
HARPER, H.J. (1939). The effect of natural gas on the growth of micro-organisms and the accumulation of nitrogen and organic matter in the soil. Soil Science 48, 4 6 1 - 4 6 6 .
HAZEU, W. and STEENNIS, P. J. (1970). Isolation and characterization of two vibrio-shaped methane-oxidizing bacteria. Antonie van Leeuwenhoek 36, 6 7 - 7 2 .
HERBERT, D., PHIPPS, P. J. and STRANGE, R. E. (1971). Chemical analysis of microbial cells. In
Methods in Microbiology, vol. 5B, pp. 209-344. London and New York: Academic Press.
HILL, S. (1971). Influence of oxygen concentrations on the colony type of Derxia gummosa grown
on nitrogen-free media. Journal of General Microbiology 67, 7 7 - 8 3 .
LARGE, P.J. and QUAYLE, J.R. (1962). Microbial growth on CI compounds. V. Enzyme activities in
extracts of Pseudomonas AMI. Biochemical Journal 87, 386-396.
LAWRENCE, A. J. and QUAYLE, J. R. (1970). Alternative carbon assimilation pathways in methaneutilizing bacteria. Journal of General Microbiology 63, 371-374.
NAMSARAEV, B. B. and ZAVARZIN, T. A. (1972). Trophic relationships in a methane-oxidizing
culture. In Mikrobiologiya 4 1 , pp. 999-1006, English translation pp. 887-892. New York:
Plenum Publishing Corp.
PAREJKO, R. A. and WILSON, P. W. (1971). Kinetic studies on Klebsiella pneumoniae nitrogenase.
Proceedings of the National Academy of Sciences of the United States of America 68, 2016-2018.
POSTGATE, R. (1971). Biochemical and physiological studies with free-living, nitrogen-fixing bacteria. Plant and Soil, Special Volume 1971, 551-559.
QUAYLE, J. R. (1972). The metabolism of one-carbon compounds by microorganisms. Advances in
Microbial Physiology 7, 119-203.
SCHOLLENBERGER, C. J. (1930). Effect of leaking natural gas upon the soil. Soil Science 29,
261-266.
WHITTENBURY, R., PHILLIPS, K. C. and WILKINSON, J. F. (1970). Enrichment, isolation and
some properties of methane-utilizing bacteria.Journal of General Microbiology 61, 205-218.
16
INVALIDITY OFTHEACETYLENE REDUCTIONASSAYIN
ALKANE-UTILIZING,NITROGEN-FIXING BACTERIA
ByJ. A.M.DEBONT and E.G.MULDER
Applied and Environmental Microbiology, 31(1976) 640-647
The causeof the failure of the C 2 H 2-C2H4 assay for nitrogen-fixing bacteria growing on loweralkaneswasstudied. Acetylene wasa strong competitive
inhibitor of methane oxidation for methane-utilizing bacteria, as well as for
the oxidation of lower alkanesby other bacteria, sothat energy and reducing
power were no longer available for the reduction of acetylene by nitrogenase.
Nitrogen-fixing bacteria grown on alkanes may reduce acetylene when intermediates of alkane-breakdown or other substrates oxidizable in the presence
of acetylene are supplied. Ethylene co-oxidation is not responsible for the
failure of the test,because acetylene also inhibits this co-oxidation along with
methane oxidation.
INTRODUCTION
Because of its sensitivity and simplicity, the acetylene reduction assay is generally
applied to measure biological nitrogen fixation (7). In using the C2H2-C2H4 assay, it is
assumed that (i)acetylene doesnot interfere with other metabolic activities of the system
under study and (ii) ethylene is stable during the investigation. These two factors have
not been studied thoroughly as yet, although reports have appeared suggesting that they
may influence the resultsof the test. Brouzesand Knowles(1)reported the prevention of
the normalgrowth pattern of anitrogenase-repressed culture of Clostridiumpasteurianum
in a medium supplemented with (NH 4 ) 2 S0 4 with 0.1 atmosphere of acetylene in the gas
phase. The acetylene inhibited cell proliferation and prevented an increase inthe rate of
carbon dioxide production,normally associated with growth.
The C 2 H 2 -C 2 H 4 test failed to show nitrogenase activity in nitrogen-fixing, methaneoxidizing bacteria of strain 41 of the Methylosinus type (2). Nitrogenase activity could
not be assayed by the test when the bacterium wasgrowingon methane,although 1S N 2
was fixed. Bacteria growing on methane co-oxidized ethylene, but cells growing on
methanol did not. This co-oxidation of ethylene by methane-grown cells might have
caused the test to fail. Alternatively, acetylene might have blocked methane oxidation,
thus preventing asupply of energy and reducing power to the nitrogenase.
This paper gives the results of a more detailed study of acetylene inhibition of cell
metabolism in the C2H2-C2H4 assayin alkane-utilizing bacteria.
17
MATERIALSANDMETHODS
Organisms. Mycobacterium vaccae,originally isolated by Ooyama and Foster (11),
was provided by J.J.Perry, North Carolina State University, Raleigh. Strains 41 and E20
havebeen described previously (2,3).
Strain Et32 was isolated by incubating 10g of soil with 25 ml of mineral salts (MS)
medium (see below) under 10%ethane in air ina 1-literErlenmeyer flask. After 2weeks
of incubation, the enrichment was streaked on platesof MSmedium.The bacterium was
isolated after incubating under ethane in air, followed by restreaking on the same
medium.Strain Et32 isagram-negative,yellow-pigmented rod.
Strain H2 was similarly isolated from soil, except that hexane replaced ethane. Strain
H2isagram-positive pleomorphic bacterium.
Strain 3b was isolated from garden soil incubated in an Erlenmeyer flask containing
10%ethane in air. Every 3weeks the flask was flushed with air, and the ethane concentration was restored. After 4months, a macroscopically visible, yellowish colony had
appeared on the surface of the soil sample. Material from this colony was streaked on
plates of nitrate-free MS medium. Colonies had grown on these plates after 6weeks of
incubation in an ethane-containing desiccator at reduced oxygen pressure.Apure culture
wasobtained after restreaking on the same medium.
Strain HI2 was isolated by incubating amixture of different soil and water samplesof
approximately 10g with 25 ml of nitrate-free MS medium in asealed 2-liter Erlenmeyer
flask. Hexane (2 ml) was added, and the oxygen pressure was reduced by flushing with
nitrogen. Material from the enrichment wasstreaked on platesof nitrate-free MSmedium
after 40 days of incubation. After 4 weeks of incubation in a desiccator containing 5%
oxygen and hexane, colonies on these plates were well developed. The pure culture was
obtained after restreaking on the same medium.
Medium. The MS medium, used throughout the investigation, containing the following salts in 1liter of deionized water: NaN0 3 , 2.0g;K 2 HP0 4 , 0.5 g;KH 2 P0 4 , 0.5 g;
MgS0 4 -7H 2 0, 0.2 g;CaCl2,0.015 g;FeS0 4 -7H 2 0, 0.001 g;CuS0 4 5H 2 0, 5jug;H 3 B0 3 ,
10^g; ZnS0 4 -7H 2 0, 70/ig; MnS0 4 -5H 2 0, 10jug; Na 2 Mo0 4 -2H 2 0, 100Mg; final pH,
6.8.For solid media, 1.5%agarwasadded to this nutrient solution.
Chemicals. Methane, ultrapure (99.97%), and other gaseous substrates (commercial
purity)wereobtained from the Matheson Co.
Culture conditions. Weekly subcultures on slants of MS medium or nitrate-free MS
medium were kept in desiccators at reduced oxygen pressure (5%or less) with approximately 5% of the desired gas and N2 present at 30C. Growth experiments were also
carried out at this temperature.
Oxygen uptake. Dissolved oxygen concentrations were measured at 30 Cwith aYellow Springs Instruments model 53oxygen monitor equipped witha polarographic sensor.
This electrode fits into a 15-mlcell;it hasanaccessslot for removal of overflowing liquid.
The slot can also be used for addingorwithdrawing smallsamples from the cell.
Gas chromatorgraphicanalyses. Analyses were carried out with a flame-ionization
18
detector with a Becker type 409 or type 417 chromatograph. A Porapak R column
(110 cm by 4 mm) with nitrogen as carrier gas was used for assaying methane (50 or
150C when in solution), acetylene reduction (50 C), ethylene and ethane (80 C), and
methanol (120 C). Acetylene wasassayed with aPorapak T column (120 cmby 4mm)at
90C.
RESULTS
Methane-oxidizing bacterium strain 41 of the Methylosinus type. The methane-oxidizing,nitrogen-fixing strain 41 did not accumulate ethylene when exposed to methane in
the presence of acetylene. However, ethylene was produced linearly by methane-grown
cells when methanol or formate was added during the assay (Fig. 1). Also, compounds
other than methanol or formate, which are both intermediates of methane oxidation,
supported the reduction of acetylene,e.g., ethanol and butanol.
Jf co-oxidation of ethylene by methane-grown cells caused the failure of the test,
ethylene should not be accumulated by such cells in the presence of methanol or formate
unless these compounds specifically suppressthe co-oxidation. Addition of methanol toa
50
Time(min)
100
Fig. 1. Effect of methanol and formate on acetylene reduction by strain 41 grown on
methane in nitrate-free MSmedium. Portions (9ml) of a 100-ml culture, growing logarithmically in a 1,000-ml Erlenmeyer flask, wereinjected into 100-ml Erlenmeyer flasks
containing 10% CH4> 5% 0 2 , 10%C 2 H 2 , and 75%N2 with 1ml of nitrate-free MS
medium containing 0.1% of the compounds indicated. Symbols:(•) Methanol;(o)sodiumformate; (•) no supplementary compound added.
19
50
additionof
methanol
x
\
\
£
d
d
25
1.25
V
*
X
\
\
\
\\
6
a
.e
50
Time(min)
Fig.2. Inhibition by methanol of methane oxidation and of ethylene co-oxidation. A
100-ml culture of strain 41, grown on methane in MS medium, was centrifuged and
suspended in 10ml of 0.03 Msodium phosphate buffer, pH6.8. This suspension was
injected into a 100-ml Erlenmeyer flask, containing methane and ethylene in air. Methanol was injected at the time indicated. Symbols: (•) Methanol (liquid phase); (o)
ethylene (gasphase);(x)methane (gasphase).
cell suspension reduced the rate of ethylene co-oxidation aswell as the rate of methane
oxidation, but the co-oxidation was not completely stopped by methanol (Fig.2),suggesting that co-oxidation was not responsible for the failure of the test with methanegrown cells.
Acetylene, always present during the assay, suppressed the oxidation of methane as
well as the co-oxidation of ethylene (Fig.3).Thisobservation definitely rules out co-oxidation of ethylene asthe possible causeof the failure of the C2H2-C2H4 assay.
Mechanism of inhibition of methane oxidation by acetylene. When strain 41 was
cultivated on methane in MS medium, the growth of the bacterium was completely
inhibited in the presence of 10%acetylene, whereas growth on methanol in MSmedium
was not affected. Apparently, the first step in the degradation route of methane, the
oxidation of methane to methanol,wasblocked by acetylene.Thiswasalsoshown by the
course of the oxygen uptake of whole-cell suspensions of strain41 grown on methane
20
(Fig.4). Acetylene suppressed methane-dependent oxygen uptake, whereas it did not
influence methanol-dependent oxygen uptake.
The first step in the breakdown of methane by strain 41 isperformed by an adaptive
enzyme system. Methane did not increase oxygen uptake of whole-cell suspensions of
methanol-grown bacteria (Table 1). The enzyme system involved in methane oxidation is
presumably analogous to the methane hydroxylase described for Methylococcuscapsulatus (12) and for Pseudomonasmethanica (5). Methane hydroxylase is also capable of
oxidizing ethane (12) and carbon monoxide (5), explaining the adaptive nature of the
co-oxidation of these compounds by strain 41. The co-oxidation of ethylene was also
adaptive, demonstrating that methane hydroxylase probably oxidized this compound.
Oxidation of methane and co-oxidation of methane hydroxylase-dependent cosubstrates
<.
T.
1.5
addition of addition of
acety lene
ethyl sne
\^ \ '
>I
100
K
*
a.
V ^ _
o
0
1
>>
\
50 I
-O—-O-0
D--0
X,
"--X-X--X--X
-
1
25
1
50
Time(min)
1
75
Fig.3. Inhibition by acetylene of methane oxidation and of ethylene co-oxidation.A
100-ml culture of strain 41, grown on methane in MS medium, was centrifuged and
suspended in 10ml of 0.03M sodium phosphate buffer, pH6.8. This suspension was
injected into a 100-ml Erlenmeyer flask, containing methane in air. At the time indicated, 1.5 ml of approximately 5,000jul of C2H4 per liter in air and 2ml of approximately 750julof C2H2 per liter in air were injected into the flask. Symbols: (•) Methane;(o)ethylene;(x)acetylene.
21
buffer CH4
Airsaturated
*
0
i
CH 4C, H,.CH4 CH 3OH C2 H,
*i
10
Time (min)
*
v
*
20
Fig. 4. Recorder trace of the uptake of dissolved oxygen by a whole-cell suspension of
strain 41. A culture growing logarithmically on CH„ in MS medium was centrifuged and
suspended in an equal volume of 0.03 Msodium phosphate buffer, pH 6.8. At the times
indicated by the arrows, 0.05 ml of the same buffer, 0.05 ml of buffer saturated with
CH„ or with C 2 H 2 , or 0.05 ml of buffer with 0.1% CH 3 OH was injected into the
suspension.
15
Time (min)
Fig. 5. Uptake of acetylene by strain 41 grown on CH4 or on CH 3 OH. Cultures of
250 ml were centrifuged and suspended in 10 ml of 0.03 M sodium phosphate buffer,
pH 6.8. The suspensions were injected into 100-ml Erlenmeyer flasks containing C 2 H 2 in
air. Methane-grown cells in the presence (x) or absence (•) of CH 4 ; concentration of
CH 4 (o); methanol-grown cells (A).
22
Table 1. Rates of oxygen uptakeby cellsuspensionsof strain 41 a
CH3OH grown
CH4 grown
Substrate
Methane
Ethane
Ethylene
Acetylene
Carbon monoxide
Methanol
Ethanol
Formate
Formaldehyde
-C 2 H 2
+C2H2
100
100
100
0
0
0
0
0
1,300
800
250
600
50
1,300
800
250
600
-C 2 H 2
+C2H2
0
0
0
0
0
500
400
100
500
500
400
100
500
a Cultures of strain 41 pregrown on methane (CH4 grown) or methanol (CH3OH
grown) werecentrifuged and suspended in0.03 M sodium phosphate buffer, pH6.8.The
course of the oxygen uptake was recorded for 5 min with an oxygen electrode with
(+C2H2) and without (-C 2H2) acetylene at afinal concentration of 1%.Substrateswere
added by injection into the suspension phosphate buffer with 1%of substrate dissolved
(liquid and solid substrates) or saturated with 100%of thegases.Ratesof oxygen uptake
are expressed as percentages of increase in oxygen uptake over the endogenous rate.
were inhibited by acetylene. Oxidation of intermediates of the methane breakdown (e.g.,
methanol), as well as that of cosubstrates not dependent upon the hydroxylase for their
breakdown (e.g., ethanol), was not influenced by acetylene (Table 1). Acetylene itself
was apparently not co-oxidized by the hydroxylase, asjudged by the oxygen uptake data.
However, by applying lower concentrations of acetylene, its disappearance from the gas
phase could be directly measured, and then the effect of methane-grown cellson acetylene was assessed. Methanol-grown cells did not show uptake of acetylene,indicating that
methane hydroxylase wasinvolved in the removal of acetylene from thegasphase(Fig.5).
Activity of methane hydroxylase is obtained only with difficulty in cell-free extracts
and, moreover, it does not remain stable for long (5, 12). Therefore, experiments with
whole-cell suspensions were undertaken to study the inhibition of methane hydroxylase
activity by acetylene. Uptake of methane and of dissolved oxygen, both dependent on
methane concentration, by wholecell suspensionsof strain 41wasmeasuredwith anoxygen-electrode cell.
The rate of oxygen uptake, after correcting for endogenous respiration, was directly
proportional to the rate of methane uptake but independent of the actual concentration
of methane.An apparentKm of approximately 1.7%for methane in the gasphase at 30C
was obtained both by measuring the uptake of methane and by recording the rate of
oxygen uptake as a function of the actualmethane concentration (Fig.6a,c,and e).The
rate of oxygen uptake was independent of the oxygen concentration (Fig.6d). The
inhibition of oxygen uptake after the addition of acetylene was taken as a measure of
the inhibition of methane oxidation. Due to the uptake of acetylene,only the deviation
23
a
X<
VI
G
3
u
o
u .o
ps ed
25
50
(Time (min)
1/%CH,
S
(c)
I Dissolved 8oxygen o-
air
air
saturated
T3 > 0>
1s
L
1
a°
1
25
50
Time(min)
10
Time(min)
75
\l
additionof
acetylene
air
saturated
V
(0
S°
1
i
25
50
Time (min)
c
50
5 eo«
(h)
•a x c
c
o =
u
"« ><
p
^-'
i)
° •*•*
2 •"
is 25
"*-* S- Q, tH
^t>'
2 U a-S
""
0.5
1.0
1/%CH4
24
i
r
-*-^*"""^
^^^^^"""^
-***
0
-""^"''""'^
1
2
Acetylene(p.p.m.)
during the first 3min after acetylene addition could be used for the purpose of this
experiment (Fig.6f). The result of eight measurements of inhibition of methane-dependent oxygen uptake by two different concentrations of acetylene at varying methane
concentrations is shown in Fig.6g.The graph obtained is consistent with a competitive
pattern of inhibition. An apparent Km of 0.5 jzl/liter for acetylene in the gas phase at
30 Ccanbe decuded from Fig.6h.
Other alkane-utilizing bacteria. Inhibition of growth on methane by acetylene was
not restricted to strain 41. Fifteen other strains of methane-oxidizing bacteria isolated
from soil and water, includingMethylosinus as well asMethylomonas types, were cultivated on slants of MSmedium with 10%methane in desiccators with andwithout 50jul
of acetylene per liter.The strainsgrewonlyinthe absence of acetylene.
The study of the inhibition of cell metabolism by acetylene has been extended to
bacteria utilizing straight-chain hydrocarbons. Five such bacteria were isolated from soil.
Two of them (strain 3b and HI2) were capable of fixing atmospheric nitrogen. Except
strain H12, growth at the expense of lower hydrocarbons was prevented by acetylene.
With hexadecane and decane, or with nonhydrocarbon substrates, acetylene did not
influence growth (Table 2).
Because the oxidation of ethane by strain 3b was inhibited by acetylene, no appreciable ethylene formation from acetylene was found with ethane as substrate. After the
addition of acetaldehyde, presumably an intermediary product in the breakdown of
ethane, ethylene was formed (Fig.7). Strain H12 actively reduced acetylene with butane
as its energy source, ascould be anticipated from its capacity to grow onthis substrate in
the presence of acetylene.
Fig.6. Inhibition of methane oxidation by acetylene. Aculture of strain 41 piegrown
on methane in MSmedium was centrifuged and suspended in0.03 Msodium phosphate
buffer, pH6.8. Approximately 0.5 ml of the suspension was placed in the oxygenelectrode cell together with appropriate amounts of buffer solution saturated withCH4
or 0 2 to give the desired concentrations of the gases in the solution. The final volume
was brought to 10mlwith buffer solution.Concentrations of CH4 andC2H2 insolution
are expressed as concentrations in the atmosphere in equilibrium with the solution.
Methane concentration in the liquid phase was followed by periodically taking 2.5-M1
samples from the cell for gas chromatographic analyses (a). From this progress curve,
tangents at different CH4 concentrations wereusedto obtain aIineweaver-Burk reciprocal plot (b). The same procedure was used to find the apparent K m for methane using
the oxygen-electrode trace after correcting for endogenous respiration (c). The actual
CH4 concentrations needed for the reciprocal plot (e) were known from the methane
progress curve (a). Independence of CH4 oxidation of 0 2 concentration was demonstrated with a suspension that was not limited in methane (d). Inhibition by C2H2 of
CH4-dependent 0 2 uptake was found by injecting buffer solution in equilibrium with
1,000julof C2H2 per liter into the cell, (f) shows the oxygen traceasaffected by 30 M1
of C2H2-containing buffer; the arrow indicates the addition of C 2 H 2 . The solid line in
(g) represents the result obtained in (e).Taking this lineasastandard,pointshavebeen
calculated by measuring the slope of the oxygen trace before and after the addition of
10n\ (o) or 20jul (x) of C2H2-containing buffer at different CH4 concentrations.
Replotting valuesof (g)for 1%CH4 (o)and 2%CH4 (•) produces(h).
25
200
Time(min)
300
Fig. 7. Acetylene reduction by strain 3b as affected by ethane and acetaldehyde. Portions (10ml) of a culture growing on ethane in nitrate-free MSmedium were injected
into 100-ml Erlenmeyer flasks containing 10%C 2 H 2 , 2%0 2 , 10%C2H6, and 78%N2
(o); (x) C2H6 omitted, 88%N 2 ; (•) 5 M' of acetaldehyde injected as indicated by the
arrow.
Table 2.Inhibition ofgrowthof hydrocarbon-utilizingbacteriabyacetylene*
Growth substrates
Organism
Methane
Ethane
a?
a" a" a
u + i
i
+
Propane
i
41
M. vaccae
Et32
H2
E20
3b
H12
Butane
a" af
u i
a
+
Hexane
a
u
Decane
a" a
i
i
+
Hexadecane
Methanol
a£ a" a" a
u
i
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
a" a
u
i
+
+
+
+
+
+
+
a Organisms were grown on the respective substrates and streaked onto slantsofMS
medium and slants of a yeast extract-glucose medium (YEG). The slantswereplacedin
sealed 1-liter Erlenmeyer flasks in the absence (-C 2 H 2 ) or presence (+C2H2) of 10%.
acetylene. Substrates were given to the MS medium slants at concentrations of10%
(gases), as 10 ml of a 1%solution in water in the flasks (alcohols),orbyputting adrop
of the substrate onto the slants (higher alkanes). Growth (+) or no growth (-) was
recorded after 3weeksof incubation.
26
YEG
a" a]
u"
i
+
+
+
Ethanol
+
+
+
+
+
+
+
+
+
+
DISCUSSION
Co-oxidation of ethylene does not cause the failure of the C2H2-C2H4 test with the
nitrogen-fixing strain 41 of the Methylosinus type when acetylene issupplied with methane as the only carbon and energy source. Co-oxidation of ethylene is completely prevented in the presence of acetylene. Acetylene not only completely preventsthe co-oxidation of ethylene, but it also inhibits very strongly the oxidation of methane.Thusthe
supply of energy and reducing power to the nitrogenase, needed for the reduction of
acetylene, is impeded. Moreover, if no substrate is availableto thebacterium, the nitrogenase presumably is insufficiently protected from oxygen. Thebacterium may overcome
these obstacles when it is given methanol or other substrates that are oxidized in the
presence of acetylene. Results supporting these conclusions have recently been reported
by Whittenbury et al. (14) who also found that the useof methane asanelectron donor
in the C2H2-C2H4 test led to negative results.
Experiments with whole-cell suspensions showed that acetylene is a competitive inhibitor for methane. However, results of experiments of thistype should be treated with
caution. The physiological response of the bacterium towards different levels of methane
and acetylene was recorded. This response does not need to coincide with the action of
methane hydroxylase since, apart from methane, the enzyme depends upon oxygen and
presumably nicotinamide adenine dinucleotide, reduced form (NADH), for its catalytic
activity. The level of oxygen does not interfere with the physiological response to methane oxidation. The actual NADH level in the bacterial cell during the experiments is
unknown but will probably vary with varying methane concentrations or after the addition of acetylene. It is uncertain what influence this varying NADH level exerts on
methane hydroxylase activity, but the straight line in the Lineweaver-Burk plot, obtained
from the progress curve for methane oxidation, is encouraging. Oxygen uptake, dependent on the concentration of the substrate,hasbeen employed previously for establishing
the apparent Km for methane (8).Thevalueobtained wasapproximately 1%,which isof
the same order as the apparent Km of 1.7% found in the present investigation. If this
method of assessing an apparent Km value is acceptable, then the measurement of an
apparent Kj performed in thisway should be possible.
Acetylene is an extremely effective inhibitor of methane oxidation. The Kt value of
0.5 jul/liter in the gas phase represents a concentration as low as approximately 0.02juM
C2H2 in solution (6). Specific inhibitors of methane hydroxylase havebeen searched for
by Hubley et al.(9),but acetylene or substrates co-oxidizableby thehydroxylase (except
carbon monoxide) were not considered as likely candidates. These co-oxidizable substrates would presumably also act as competitive inhibitors for methane, albeit with a
much higher K{ than acetylene. Ethane,for instance,may inhibit thegrowth of methaneoxidizing bacteria (13).The fate of acetylene wasnot studied in the present investigation.
The situation with strain 41 and the other methane-oxidizing bacteria tested wasalsomet
with the bacteria growing on lower hydrocarbons. Only the enzyme system involved in
the first attack on the hydrocarbon was inhibited by acetylene. Growth upon long-chain
27
hydrocarbons was not inhibited by acetylene, suggestingthat the inhibition ofthe alkane
hydroxylase by acetylene depends on the size of the substrate molecule. This indicates
the existence of a direct relation between substrate and inhibitor with respect to the
hydroxylase. The type of inhibition is probably the same asthat found for the methaneoxidizing bacteria. Strain H12 was an exception, because it wasnot inhibited by acetylene,evenwhen it wasgrowingon butane.
The experiments show that the C2H2-C2H4 test cannot be employed for measuring
nitrogenase activity in methane-utilizing bacteria or in bacteria utilizing lower hydrocarbons when alkanes are the sole energy source. Nitrogenase activity can be measured
by growing these bacteria on carbon sources other than the alkanes. However, this may
raise a problem with methane-oxidizing bacteria sincemethanol,the only alternative carbon source, is toxic to some strains when supplied at substrate-level concentrations (13).
Nitrogenase activity of methane-grown cells can only be measured by supplying the
organism during the test with a different energy source, but the linear rate of ethylene
production was not always reproducible. Furthermore, in this way it is impossible to
measure nitrogenase activity quantitatively.
In natural habitats, the contribution to the fixation of atmospheric nitrogen by methane-oxidizing bacteria and bacteria using lower hydrocarbons is overlooked when using
the C2H2-C2H4 test. Preliminary experiments in which small amounts of methanol or
formate were added to samples from various habitats to reveal possible nitrogen fixation
by methane-oxidizing bacteria were not successful. Scaling up the activity of theseorganisms by incubating water samples under methane and then applying the test with methanol or formate added has given inconsistent results. As a consequence, nitrogenase
activity should not be measured with the C2H2-C2H4 test in natural habitats where
bacteria utilizing methane or lower hydrocarbons are present. Nitrogenase activity could
be measured with 1SN 2 .
The strong effect of acetylene upon the metabolic activities of the bacteria studied
calls for a careful approach when employing the C2H2-C2H4 test. Other metabolic activities already found to be affected byacetylene include the production of methane (4, 10)
and the growthofC. pastewianum (1).
ACKNOWLEDGMENT
Support for the research has come from grants of the VEGGasinstituut and the N.V.
Nederlandse Gasunie.
LITERATURECITED
BROUZES, R., and R. KNOWLES. 1971.Inhibition of growth of Clostridium pasteurianum by
acetylene:implication for nitrogen fixing assay.CanJ. Microbiol. 17: 1483-1489.
28
2. DE BONT, J. A. M.,and E. G. MULDER. 1974. Nitrogenfixation and co-oxidation of ethylene
byamethane-utilizing bacterium. J.Gen.Microbiol.83: 113-121.
3. DE BONT, J. A. M. 1975. Oxidation of ethylene by bacteria. Ann. Appl. Biol. 81: 119-121.
4. ELLEWAY,R.F.,J. R. SABINE,and D.J.D.NICHOLAS. 1971.Acetylene reduction by rumen
microflora. Arch.Microbiol. 76: 277-291.
5. FERENCI, T. 1974.Carbon monoxide-stimulated respiration in methane-utilizing bacteria.FEBS
Lett.41: 94-98.
6. GUERIN,H. 1952.Traitedemanipulation et d'analyse desgaz.Masson et Cie, Paris.
7. HARDY, R. W. F., R. C. BURNS, and R. D. HOLSTEN. 1973. Applications of the acetyleneethyleneassayfor measurement ofnitrogen fixation. SoilBiol.Biochem.5: 47-81.
8. HARRISON, D. E. F. 1973. Studies on the affinity of methanol-and methane-utilizing bacteria
for their carbon substrates.J. Appl.Bacteriol, 36: 301-308.
9. HUBLEY, J. H., A.W. THOMSON,and J. F.WILKINSON. 1975.Specific inhibitors of methane
oxidation inMethylosinustrichosporium. Arch.Microbiol. 102: 199-202.
10. MACGREGOR, A. N.,and D. R. KEENEY. 1973.Methane formation by lake sediments during
invitro incubation. Water Resour. Bull.9: 1153-1158.
11. OOYAMA, J., and J. W.FOSTER. 1965. Bacterial oxidation of cycloparafinnic hydrocarbons.
Antonievan Leeuwenhoek;J. Microbiol. Serol.31: 45-65.
12. RIBBONS, D. W., and J. L. MICHALOVER. 1970. Methane oxidation by cell-free extracts of
Methylococcuscapsulatus. FEBSLett.11: 41-44.
13. WHITTENBURY, R., K. C. PHILLIPS,and J. F. WILKINSON. 1970.Enrichment,isolation and
somepropertiesof methane-utilizing bacteria.J.Gen.Microbiol.61: 205-218.
14. WHITTENBURY, R., H. DALTON, M. ECCLESTON, and H. L. REED. 1975.Thedifferent types of methane oxidizing bacteria and some of their more unusual properties, p. 1-9. In Proc.
Int. Symp.onmicrobialgrowth onCy-compounds,Tokyo. Society of Fermentation Technology,
Tokyo.
29
NITROGENFIXATION BYMETHANE-UTILIZING BACTERIA
ByJ. A.M.DEBONT
Antonievan Leeuwenhoek42 (1976) 245-254
Several pure cultures of methane-utilizing bacteria,including types Iand II
membrane representatives, were found to be capable of fixing nitrogen. One
nitrogen-fixing isolate grew in liquid medium, but not on a solid agar medium. Apparently, the ability to fix nitrogen is common in methane-oxidizing
bacteria.
INTRODUCTION
The fixation of nitrogen by a pure culture of the methane-oxidizing bacterium
strain41 of the Methylosinus type has been demonstrated with 15 N 2 by de Bont and
Mulder (1974). The bacterium was very sensitive to oxygen when dependent upon N2 as
the source ofnitrogen,aconsequence ofthe nitrogenase's sensitivity towards oxygen.
When measured by the acetylene-reduction assay, the nitrogenase activity of thebacterium was erratic. Ethylene was formed from acetylene when the bacterium wasgrown
in anitrogen-free medium and methanol served asthe substrate. There wasno appreciable
reduction of acetylene when methane was the source of carbon and energy, probably
because the acetylene competitively inhibited the oxidation of methane in this bacterium
(de Bont and Mulder, 1976). However, when substrates were available that were oxidizable regardless of whether acetylene was present, the activity of the nitrogenase in methane-grown cellswasnot affected by acetylene.
The present study surveyed the nitrogen-fixing capacity of methane-oxidizing bacteria.
MATERIALSANDMETHODS
Culture medium. The mineral-salts solution (MS medium) used in this study contained the following salts per liter of deionized water: NaN0 3 , 2.0g; K2HPO4, 0.5g
KH 2 P0 4 , 0.5g; MgS0 4 -7H 2 0, 0.2 g; CaCl2, 0.015g; FeS0 4 -7H 2 0, 0.001g
CuS0 4 -5H 2 0, 5/ug; H3BO3, 10/ug; ZnS0 4 -7H 2 0, 70tig; MnS0 4 -5H 2 0, lOjug
Na 2 Mo0 4 -2H 2 0, 100fig;the final pH was 6.8. For solid media 1.5% agar wasadded to
this nutrient solution.
31
Oiemicals. Ultra pure (99.97%) methane (Matheson Co., East Rutherford, N.J.,
USA)wasused.
Bacteria. Strain XX(Patt et al., 1974),strain 1G(Adamse et al., 1972),strain 41 (de
Bont and Mulder, 1974), and strains N3A, 3.2B2, D2, 1, NIG 3A, NIDI, and ROSE
(Hazeu, 1975) were used.
Culture conditions. Weekly subcultures on slants of MSmedium or nitrate-free MS
medium were kept at 30 Cin desiccators with lowered oxygen pressure with about 5%
methane. Cultures growing in 25-ml tubes with 10mlofliquid medium were alsokept in
desiccators.
Gas chromatography. A Becker model 409 or model 417 chromatograph with a
flame ionization detector was used with a 110cm x 4 mm Porapak Rcolumn with
nitrogen as the carrier gas at 50Cto assay for methane and acetylene reduction. Oxygen
and methane were measured by thermal conductivity using a 200 cm x 4 mm column
with a 13Xmolecular sieve(60-80 mesh)andnitrogen asthe carriergas.
Acetylene reduction. The reduction of acetylene was assayed after quickly replacing
with a suba seal the cotton plugs of the 25-ml tubes in which cultures were grown on
slants or in liquid methane-containing desiccators. A methanol or formate solution
wasinjected prior to the addition of 2mlof acetylene.
Electron microscopy. Strain 41 and strain 3 cells collected from mid-exponential
phase cultures by centrifugation and washed in S0rensen buffer pH 7.0 were prefixed in
0.5% 0 s 0 4 for 15min, fixed overnight in 1%OSO4 and washed in Kellenberger buffer.
The material was transferred into agarblocks,stained with uranyl acetate for 1hour, and
embedded in Epon-812.
Strain K cells, embedded in agar microcapsules,were treated with Palade's fixative for
4hours at pH7.4 at 4 C, dehydrated in acetone, and embedded in Epon-812. Sections
were stained withuranyl acetate for 30min and withlead citrate for 1min.
RESULTS
Enrichment and isolation. About 1gof inoculum was incubated in 10mlof anitrogen-free MS medium to which 50mg/liter of yeast extract was added. Nine soilsamples
from variouslocations andtwo soilsamplesthat had been exposed to natural gasescaping
from pipe lines, seven water samples from ditches and rivers, saliva of a cow, decaying
leaves, and decaying straw from a dung hill were used. Theinoculated medium waskept
in a 100-ml Erlenmeyer flask with an atmosphere of 5%oxygen, 5%methane, and 90%
nitrogen gas. The presence of oxygen and methane wasmonitored weekly, and whenever
needed, these gases were refurnished. Growth appeared inthe flasks after 2to 6weeksof
incubation. Then,adrop of the turbid medium wastransferred to fresh medium and after
incubating this under the same conditions, the material was streaked onto plates of
nitrogen-free MS medium; some plates contained medium to which 50mg/liter yeast
extract had been added.
32
0 •»'
*
»
'K
Ail
%/
• . •
^ •
— • * A
> *
'
>2
•• I \
1
4k
'\
*.*
i
#
\
!K
%
. *
I
* • • - <
%
4
Fig. 1. Strain 41 of a Methylosinus sp. from a 10-day culture grown on slants of MS
medium with methane.Thebar represents 10 Mm.
Fig.2. Strain 2 of a Methylomonas sp. from a 3-day culture grown on slants of MS
medium with methane.Thebar represents 10Mm.
Fig. 3. Strain 16 from a 5-day culture grown on slants of MS medium with methane.
Thebarrepresents 10 Mm.
Fig.4. Strain Kfrom a 10-day culture grownin liquid MSmedium on methane.Thebar
represents 10 Mm.
33
The plates were incubated for up to 2months in desiccators with an atmosphere.of
about 5% oxygen, 5% methane, and 90% nitrogen. Colonies were selected at different
time intervals and streaked to purity.
Although good growth appeared in the liquid medium with all samples, isolation of
the nitrogen-fixing methane-oxidizing bacteria was difficult. Colonies of methane-oxidizing bacteria readily appeared on plates, but the bacteria were often lost on further
purification by restreaking.
Fig.5. Thinsection of strain 41 of aMethylosinussp.grown in nitrogen-free MS medium on methane, showing the type IImembrane system.Thebar represents 0.25jum.
Fig.6. Thin section of strain 2 of aMethylomonas sp.growninnitrogen-free MS medium on methane,showing the type 1membrane system.Thebar represents 0.25,um.
34
Table 1. Methane-oxidizing bacteria counted in different sourcesof inoculum.
Source of inoculum
Mostprobable number
Nitrate-free medium
Ditch water (a)
Mud of ditch (a)
Ditch water (b)
Mud of ditch (b)
Sandy soil
Clay
350
130 000
240
70 000
13 000
11000
Nitrate-containing medium
700
110000
540
13000
13000
17000
Samples taken in winter time from ditch water, the aerobic upper mud layers of
ditches, and soil, were serially diluted (1:10) in nitrogen-free MSmedium.Tubes (5per
dilution) containing 9ml of nitrogen-free MSmedium and tubes containing 9 mlofMS
medium were incubated in desiccators containing an atmosphere of approximately 5%
oxygen, 5%methane, and 90%nitrogen.Thedesiccators wereopened weekly to remove
tubes showing growth. The number of positive tubes did not increase after seven weeks
of incubation. Numbersare calculated perg.
Only three strains were isolated from the nine soil samples while no pure culture was
obtained from the dung hill sample. The bacteria isolated from the leaves,from the cow
saliva, from one of the two soil samples that had been exposed to natural gas,and seven
isolates from the water samples all closely resembled the three soil isolates. All these
strains were motile in young cultures and formed exospores upon aging,but the cellsize
and cell shape varied. Both straight rods and vibrioid forms were identified. They were
similar to strain 41 (Fig. 1)and were classified asMethylosinus-type bacteria according to
Whittenbury, Phillips and Wilkinson (1970).Noclear resting stage wasfound for the pink
Methylomonas type of bacteria (strains 2, 3, and 21) from three of the water samples
(Fig.2). Strain 41 had a type II membrane system (Fig. 5) as compared with the type I
membrane system of strain 2(Fig.6).
One isolate (strain 16), obtained from soil exposed to natural gas, formed no resting
stages and its growth was stimulated by the presence of yeast extract in the medium
(Fig.3).
Methane-oxidizing bacteria capableof fixing nitrogen. The occurrence of methaneoxidizing bacteria with the capacity to fix nitrogen was investigated by incubating serial
dilutions of samples in both nitrogen-free MS medium and in nitrate-containing MS
medium. The results of the most probable number (MPN) counts are given in Table 1.
Microscopic examination of the tubes used for the MPN counts revealed the presence of
Methylosinus type bacteria and the pinkMethylomonas-like organisms in the lower dilutions. The presence of nitrate in the medium did not affect the morphological characteristicsobserved microscopically.
The higher dilutions of soiland mud samples contained bacteria that differed morphologically from the two forementioned groups. Efforts to isolate these bacteria revealed
large colonies on plates of nitrogen-free MS medium streaked with material from the
higher dilutions when incubated in an atmosphere containing approximately 5%oxygen,
35
Fig.7. Colonies of strain Kgrown for 6weeksunder methane onplatesof nitrogen-free
MS medium after the first transfer from the liquid enrichment culture to the solid
medium. Subsequent transfers from these coloniesgrew only in liquid medium.
Fig. 8. Thinsectionof strain Kgrowninnitrogen-free MS medium on methane,showing
the type IImembrane system.Thebar represents 0.25 Mm.
36
5%methane, and 90%nitrogen (Fig.7). To avoidlosingthebacteria when material from
the colonies wasrestreaked, alargenumber of colonies at animmature stage was transferred directly to liquid medium. Good growth was obtained. One isolate, designated
strain K (Fig.4), isolated from a 10"6 dilution of soil, was found to be an immotile,
coccoid bacterium that had the type II membrane system (Fig. 8). This nitrogen-fixing
obligate methylotrophic bacterium grew only in liquid medium. Streaks on agar slants
did not grow.Noresting stage was observed.
Strain 1G, reported by Adamse et al. (1972) to be incapable of fixing atmospheric
nitrogen, was studied and was found to reduce acetylene when grown on methanol in
nitrogen-free MSmedium under reduced oxygen pressure. Of the seven strains of Hazeu
(1975) studied, type II strains (D2 and 1)were found to fix nitrogen asdid type Istrains
N3A and 3.2B2. No acetylene reduction could be demonstrated with type I strains
NIG 3A, NIDI, and ROSE, or with the facultative methylotrophic methane-oxidizing
bacterium strain XXof Patt et al.(1974).
DISCUSSION
Apparently, the capacity to fix atmospheric nitrogen is widespread among methaneoxidizingbacteria.Methylosinus type strains were without difficulty isolated from enrichments, and although growth was good in the liquid enrichments, other types of methane
oxidizers could not easily be isolated. This was also the case with material from higher
dilutions of soil and mud samples. The coccoid strains that were finally isolated did not
fit into the classification scheme of Whittenbury et al. (1970). They resembled the
coccoid strain 1of Hazeu (1975).
Although Methylomonas type strains were difficult to isolate and grow, three strains
were isolated from water. A number of other enrichments also contained these bacteria.
The pink Methylomonas type bacteria were difficult to keep alive in pure culture. Only
strain 3 survived one year of subculturing. The growth of strain 3 was enhanced in a
mixed culture with a small motile rod that frequently appeared asacontaminant of pure
cultures. The addition of traces of yeast extract to the nitrogen-free MSmedium had no
appreciable effect on the isolation of methane-oxidizing bacteria from enrichment cultures. All pure cultures, except strain 16, were isolated from plateswith yeast extract,as
well as from plates without yeast extract. The effect of yeast extract on promoting the
growth of strain 16hasnot yet been studied.
Methane-oxidizing bacteria are known to be difficult to isolate and grow, and cultivating methane-oxidizing bacteria that fix nitrogen is even more difficult because they
are sensitive to oxygen when grown without combined nitrogen. Thus,in this study only
strains that are relatively easy to isolate and to handle emerged from the enrichments.
The types which are difficult to culture in the laboratory may,nevertheless,play a more
important role in nature than might be expected from the random isolations from enrichments. Only very fewMethylosinus type bacteria were present in the mud sampleswhereasthe coccoid forms were predominant from microscopic observations.
37
Apparently, many types of methane-oxidizing bacteria can fix nitrogen under laboratory conditions and innature. Although nitrate isgenerally accepted asthe best source of
nitrogen for growing methane-oxidizing bacteria (Hazeu, 1975), in this study it was not
found to be a significantly better source than N2 when serving as the sole source of
nitrogen. Bacteria isolated from the highest dilutions of the nitrate-containing tubes were
also able to fix nitrogen. Four of the seven strains of Hazeu (1975) showed nitrogenase
activity. Whittenbury et al. (1975) demonstrated that most strains of methane-oxidizing
bacteria, previously reported to be unable to fix nitrogen (Whittenbury et al. 1970),
showed nitrogenase activity when the acetylene reduction test wasproperly used.
The results of the present study suggest that inthe soil and in the water of the ditches
investigated, the fixation of nitrogen by these bacteria is not quantitatively significant.
However, in the aerobic mud layers, where oxygen and methane come together, the
amount of nitrogen fixed by these organisms may be substantial. Particularly highactivities of methane-oxidizing bacteria havebeen recorded in the thermocline of lakes(Patt et
al., 1974; Rudd, Hamilton and Cole, 1974), where it is likely that high rates of nitrogen
fixation accompanied the rapid oxidation of methane.
Nitrogenase activity was not obtained with the facultative methylotrophic methaneoxidizing bacterium of Patt et al.(1974). Allnitrogen-fixing strainsisolated in the present
investigation were obligately methylotrophic. Rather than taking this result asanindication of the failure of facultative methylotrophic methane-oxidizing bacteria to fix dinitrogen,one might ascribe it to the isolation procedure used.
I would like to thank Miss Th. E.Werdmuller von Elgg for conducting the electron
microscopy at the Technical and Physical Engineering Research Service at Wageningen,
Professor R. S. Hanson of the University of Wisconsin (Madison, U.S.A.) for providing
strain XX, and Mr. W.Hazeu of the University of Technology of Delft (The Netherlands)
for supplying strains N3A, 3.2B2, D2, 1,NIG3A, NIDI, and ROSE. Support for the
research by grants of the VEG Gasinstituut and the N.V. Nederlandse Gasunie is gratefully acknowledged.
REFERENCES
ADAMSE, A. D., HOEKS, J., DE BONT, J. A. M. and VAN KESSEL, J. F. 1972. Microbial activities
in soil near natural gas leaks. - Arch. Mikrobiol. 83: 3 2 - 5 1 .
D2 BONT, J. A. M. and MULDER, E. G. 1974. Nitrogen fixation and co-oxidation of ethylene by a
methane-utilizing baterium. - J. Gen. Microbiol. 83: 1 1 3 - 1 2 1 .
DE BONT, J. A. M. and MULDER, E. G. 1976. Invalidity of the acetylene-reduction assay in alkaneutilizirg, nitrogen-fixing bacteria. - Appl. Environ. Microbiol. 31: 6 4 0 - 6 4 7 .
HAZEU, W. 1975. Some cultural and physiological aspects of methane-utilizing bacteria. Antonie van
Leeuwenhoek 4 1 : 121-134.
PATT, T. E., COLE, G. C , BLAND, J. and HANSON, R. S. 1974. Isolation and characterization of
bacteria that grow on methane and organic compounds as sole sources of carbon and energy. J. Bacteriol. 120: 9 5 5 - 9 6 4 .
RUDD, J. W. M., HAMILTON, R. D. and CAMPBELL, N. E. R. 1974. Measurement of microbial oxidation of methane in lake water. - Limnol. oceanogr. 19: 519-524.
38
WHITTENBURY,R., PHILLIPS,K.C. and WILKINSON,J. F. 1970. Enrichment, isolation and some
properties of methane-utilizing bacteria. — J.Gen.Microbiol.61:205-218.
WHITTENBURY, R., DALTON,H., ECCLESTON,M.and REED,H.L. 1975. Thedifferent typesof
methane oxidizing bacteria and some of their more unusual properties, p. 1-9. In "Microbial
growth on Cl-compounds", Proc. of the Int. Symp. at Tokyo, Japan, Sept. 5, 1974. - Soc. of
Fermentation Technology, Japan.
39
HYDROGENASEACTIVITY INNITROGEN-FIXING METHANEOXIDIZING BACTERIA
ByJ. A.M.DEBONT
Antonievan Leeuwenhoek 42 (1976) 255-260
Hydrogenase activity in cellsof the nitrogen-fixing methane-oxidizing bacterium strain 41 of the Methylosinus type increased markedly when growth
was dependent upon the fixation of gaseous nitrogen. A direct relationship
may exist between hydrogenase and nitrogenase inthisbacterium. Acetylene
reduction wassupported by the presence of hydrogengas.
INTRODUCTION
Hydrogen gas is associated in different wayswiththe fixation of nitrogen by bacteria.
Some nitrogen-fixing bacteria produce hydrogen gas, probably because of the activity of
the nitrogen-fixing enzyme complex (nitrogenase). Thiswasshown in work with cell-free
extracts of Azotobacter vinelandii supplied with ATP, Mg2+, and a reductant (Bulen,
Burns and LeComte, 1965).Thissystem isnot inhibited by COand isgenerally described
as the ATP-dependent H2 evolution to differentiate this reaction from the so-called
classical hydrogenase-catalyzed reaction which does not require ATP and which is inhibited by CO. Azotobacter chroococcum produced hydrogen when the cells were exposed to air containing 10%actylene and 1%CO,but nohydrogen wasproduced without
acetylene in the atmosphere (Brotonegoro, 1974).
A number of coryneform strains were shown to fix nitrogen and use hydrogen for
autotrophic growth (Gogotov and Schlegel, 1974; de Bont and Leijten, 1976.) Hydrogenase supplies energy and reductant inthese strains.
The function of hydrogenase in heterotrophic nitrogen-fixing bacteria is lessclear. In
Azotobacter species an association between hydrogenase and nitrogenase may exist
(Green and Wilson, 1953).Ahydrogenase ofMycobacteriumflavum 301 wasreported by
Biggins and Postgate (1969) to resemble that of Azotobacter which does not evolve
hydrogengas.
Competitive inhibition of N2 fixation by H2 was assessed both in intact cells (Bradbeer and Wilson, 1963) and in cell-free extracts (Hwang, Chen and Burris, 1973) of
Azotobactervinelandii.
This communication reports on the presence of hydrogenase activity in the recently
described nitrogen-fixing methane-oxidizing bacteria of the Methylosinus type (de Bont
and Mulder, 1974).
41
J&
100
Time (min)
200
Fig. 1. Effect of the source of nitrogen on the oxidation of hydrogen by strain 41
growing on methane. Suspensions (4 ml) of cultures growing logarithmically under reduced oxygen pressure in the nitrate-containing MS medium (30 Mg protein/ml) or in the
nitrate-free MS medium (35 Mgprotein/ml) were injected into silicon rubber-sealed vaccine bottles (16 ml) containing 5% 0 2 , 5% CH 4 , 1% H 2 , and 89% N 2 . After equalizing
the pressure, 0.1 ml samples were periodically withdrawn from the gas phase for gaschromatographic analyses of hydrogen and methane. The bottles were incubated at 30 C
while shaking. Hydrogen concentration above nitrate-containing (D) and nitrate-free (o)
MS medium; methane concentration above nitrate-containing (•) and nitrate-free (•) MS
medium.
MATERIALS ANDMETHODS
Bacterium and culture conditions. Strain 41 of the Methylosinus type, described
previously by de Bont and Mulder (1974) was used. The mineral salts solution (MS
medium) contained per 1 liter of deionized water: NaN0 3 , 2.0 g: K 2 HP0 4 , 0.5g
KH 2 P0 4 , 0.5g; MgS0 4 -7H 2 0, 0.2 g; CaCl2, 0.015 g; FeS0 4 -7H 2 0, 0.001g
CuS0 4 -5H 2 0, 5jug; H3BO3, lOjug; ZnS0 4 -7H 2 0, 70jug; MnS0 4 -5H 2 0, 10jug
Na 2 Mo0 4 -2H 2 0, 100fxg; final pH 6.8. Cells were grown at 30C while shakingin Erlenmeyer flasks containing an atmosphere of about 5%0 2 ,10% CH4, and 85% N 2 .
Gaschromatography. A Becker model 409 chromatograph equipped with a flameionization detector was used to assay actylene reduction at 50 C,usinga 110cm x4mm
Porapak R column with nitrogen as the carrier gas. Methane was measured on a Becker
model 406 chromatograph; oxygen and hydrogen were determined by thermal conductivity using a 300cm x 2mm Porapak Q column with nitrogen asthe carrier gasat 50C.
42
Protein assay. The Folin-Ciocalteu method was used to measure protein (Herbert,
Phipps and Strange, 1971).
RESULTSANDDISCUSSION
Attempts to cultivate the nitrogen-fixing obligate methylotroph strain 41 of the
Methylosinus type on H2 in the presence of C0 2 have not been successful. However,
hydrogenase activity in methane-growing cells could be assessed in growing cultures by
measuring the uptake of H 2 . There was some hydrogenase activity when the bacterium
was grown in nitrate-containing medium, but the enzymic activity increased markedly
when bacterial growth was dependent on the fixation of gaseous nitrogen (Fig. 1.).
Apparently, the function of hydrogenase in this strain is similar to that in Azotobacter
species and is associated with the fixation of nitrogen by the bacterium. Azotobacters
grown on N2 have a higher hydrogenase activity than ammonia-grown cells (Green and
Wilson, 1953). Three possible functions of the hydrogenase in the oxidation of H2
(presumably formed by ATP-dependent nitrogenase activity) innitrogen-fixing organisms
were considered by Dixon (1972).(1)The oxidation of hydrogen usesexcess oxygen and
helps to maintain the nitrogenase inan anaerobic environment within the cell.This could
.•
400
?a.
a.
s
t 200
B3
o
__o
01
20
Time(min)
40
Fig.2. Acetylene reduction by strain 41 with (•) and without (o) 5%hydrogen in the
atmosphere. Portions, 10ml, of a culture growing on methane in anitrate-free medium
were injected into 100-ml Erlenmeyer flasks containing 10%actylene, 5%oxygen, and
85%nitrogen before hydrogen gaswasintroduced.
43
be vital in the methane-oxidizing bacteria since oxygen wasreported to inhibit the nitrogen-fixing reaction (de Bont and Mulder, 1974). (2) The oxidation ofhydrogen prevents
nitrogenase activity from being inhibited by hydrogen. (3) Reductant and ATP are
formed upon the oxidation of hydrogen, thereby increasing the efficiency of the fixation
of nitrogen.
Because the Azotobacter hydrogenase catalyzes only the reduction of electron acceptors with H2 but does not evolve H2 (Postgate, 1974), it is unlikely that hydrogen
ions are excluded from the site of nitrogenase.
In Methylosinus strain 41, the function of nitrogenase-associated hydrogenase is unclear. Hydrogen gassupported the reduction of acetylene,indicating that its oxidation by
hydrogenase supplies reductant and energy to the nitrogenase (Fig. 2). A functional
operation of hydrogenase under these conditions is obvious, but it remains unclear
whether this mechanism explains the observed hydrogenase-nitrogenase relationship in
methane-oxidizing bacteria because the presence ofhydrogen or of nitrogenase-catalyzed,
ATP-dependent, hydrogen formation was not demonstrated during growth in nitrogenfree medium.
Support for the research is gratefully acknowledged from grants from the VEG Gasinstituut and the N.V.Nederlandse Gasunie.
REFERENCES
BIGGINS, D. R., POSTGATE, J. R. 1969. Nitrogen fixation by cultures and cell-free extracts of
Mycobacterium flavum 301. - J. Gen. Microbiol. 56: 1 8 1 - 1 9 3 .
DE BONT, J. A. M. and LEIJTEN, M.W. M. 1976. Nitrogen fixation by hydrogen-utilizing bacteria. Arch. Microbiol. 107: 235-240.
DE BONT, J. A. M. and MULDER, E. G. 1974. Nitrogen fixation and co-oxidation of ethylene by a
methane-utilizing bacterium. - J. Gen. Microbiol. 83: 1 1 3 - 1 2 1 .
BRADBEER, C. and WILSON, P.W. 1963. Inhibitors of nitrogen fixation, p. 595-614. In:
R. M. Hochster and J. H. Quastel, (eds.), Metabolic inhibitors, Vol. 2. - Academic Press,New York.
BROTONEGORO, S. 1974. Nitrogen fixation and nitrogenase activity of Azotobacter chroococcum.
- Thesis Agricultural University. Wageningen.
BULEN, W. A., BURNS, R. C. and LECOMTE, J. R. 1965. Nitrogen fixation: Hydrosulfite as electron
donor with cell-free preparations of Azotobacter vinelandii and Rhodospirillum rubrum. - Proc.
Nat. Acad. Sci. 53: 5 3 2 - 5 3 9 .
DIXON, R. O. D. 1972. Hydrogenase in legume root nodule bacteroids: Occurrence and properties. Arch. Mikrobiol. 85: 1 9 3 - 2 0 1 .
GOGOTOV, J. N. and SCHLEGEL, H. G. 1974. N 2 -fixation by chemoautotrophic hydrogen bacteria.
- Arch. Microbiol. 97: 359-362.
GREEN, M. and WILSON, P.W. 1953. Hydrogenase and nitrogenase in Azotobacter. - J. Bacteriol
65: 5 1 1 - 5 1 7 .
HERBERT, D., PHIPPS, P.J. and STRANGE, R. E. 1971. Chemical analysis of microbial cells,
p. 209-344. In: Methods in Microbiology, Vol. 5B. — Academic Press, London and New York.
HWANG, J. C , CHEN, C. H. and BURRIS, R. H. 1973. Inhibition of nitrogenase-catalyzed reductions.
- Biochim. Biophys. Acta 292: 256-270.
POSTGATE, J. 1974. Prerequisites for biological nitrogen fixation in free-living heterotrophic bacteria, p. 663-686. In A. Quispel, (ed.), The Biology of nitrogen fixation. - North-Holland Publ.
Comp., Amsterdam.
44
GENERALDISCUSSION
The results presented in paper I show that the methane-oxidizing bacterium strain 41
of the Methylosinus-type iscapable of fixing atmospheric nitrogen.Thisisthe first report
on nitrogen fixation by a pure culture of a methane-oxidizing bacterium. However, in
earlier literature there areindications that these bacteria have the capacity to fix nitrogen.
The effect of leaking natural gas upon the soil was studied by Schollenberger (1930).
A chemical analysis showed that the affected soilwashigher in replaceable ammonia than
was the soil from an adjacent area which had not been exposed to natural gas.Schollenberger did not consider bacterial oxidation of natural gas. He thought the effects of the
gas to be due to displacement of air from soil resulting in anaerobic conditions. The
notably higher concentration of exchangeable ammonia would then be the result of
biological action under these anaerobic conditions.
Harper (1930) observed a strong increase in total nitrogen and organic matter in soil
that had been exposed to natural gas.Hecollected samplesof soilsexposed to naturalgas
from leaking pipe lines and samples of normal soil taken from the samelocality. Inevery
soil that had been exposed to gas for a considerable length of time, the total nitrogen
content increased.The average nitrogen content in the soils affected by the gaswasnearly
three times that in normal soils. Oatswasplanted in soils collected near the gasleaksand
in similar soil that had not been affected by the escaping gas.The oat leaveswere darker
and more forage wasproduced by the plantsin the soil collected near the gasleak than by
those in normal soil. These observations indicated that nitrogen accumulating in soils
exposed to natural gas is available to plants. Harper realized that microorganisms were
involved in the processes he observed. He expected that hydrocarbons in the naturalgas
would supply the necessary energy, either directly or indirectly, for the growth of the
microorganisms capable of fixing nitrogen.
Comparable results were reported by Hoeks (1972). His analysis of soil showed that
the nitrogen content in soilthat had been exposed to natural gaswashigher than it wasin
normalsoil.
The two possible mechanisms for accumulation of nitrogen already suggested by
Harper (1939) were also considered by Davis, Coty and Stanley (1964). (i)The hydrocarbon is utilized by bacteria capable of fixing atmospheric nitrogen, the result being an
ultimate increase in organic carbon and nitrogen of the soil, (ii)The hydrocarbon is
utilized by microbes incapable of fixing nitrogen, but the hydrocarbon thus converted
into microbial cells ultimately becomes available to other soil microorganisms, some of
which are capable of fixing nitrogen. Davis,Coty and Stanley isolated methane-oxidizing
bacteria that were able to grow in nitrogen-free mineral salts medium. The isolated
bacteria were Gram-negative motile rods. Nitrogen fixation was measured by Kjeldahl
analysis of cultures incubated for two weeks or for two or four months. Later work
(Coty, 1967) employing 15 N 2 corroborated the Kjeldahl results. From the experimental
data it was concluded that the increase innitrogen content of soilsexposed to naturalgas
45
was probably directly due to the hydrocarbon-utilizing bacteria. However, the isolates
used in the experimentsgrewon nutrient agar,indicating that the cultureswere not pure.
Two attempts to demonstrate nitrogen fixation by methane-oxidizing bacteria using
the acetylene-ethylene test have been reported. Whittenbury, Phillips and Wilkinson
(1970) provided puzzling results.The authorsisolated aMethylosinus trichosporium from
Coty's culture and the organism wasexposed to an atmosphere containing 4.4%methane
and 1.8% acetylene for 7 or 14days. Reduction of 25% of the acetylene present was
reported, but it was not stated whether ethylene was the product formed. This is no
evidence of nitrogen fixation as the acetylene may have disappeared by co-oxidation or
by simply leaking from the incubation system.These authors also tried to grow Methylobacter strains in nitrogen-free medium because of the morphological similarity between
these strains and Azotobacter species. None, however, grew in nitrogen-free medium.
Adamse, Hoeks, de Bont and van Kessel (1972) obtained negative results with the acetylene-ethylene test even though the organism under investigation grew in a nitrogen-free
medium.
Failure of the acetylene-ethylene assay
In papersI and II an explanation is given for the erratic results obtained with the
acetylene-ethylene assay for nitrogen-fixing methane-oxidizing bacteria. When strain 41
was grown in a nitrogen-free medium with methane as substrate, the acetylene-ethylene
assay gave negative results. But when the organism was grown in the same medium with
methanol as substrate, ethylene production from acetylene was demonstrated. The apparent difference in acetylene reduction might have been due to the ability of methanegrown cells to co-oxidize ethylene. In this way, the product formed by the nitrogenasecatalysed reaction would be removed, thus masking the acetylene reduction by the
enzyme. This hypothesis was supported by the observation that ethylene was not cooxidized by cellsgrown on methanol. However, the hypothesis had to be givenupwhen it
was found that co-oxidation of ethylene by methane-grown cells was completely inhibited by acetylene. Acetylene not only prevented the co-oxidation of ethylene, but it
also inhibited very strongly the oxidation of methane. This result explains why the
bacteria cannot reduce acetylene whenmethane isgiven asoxidizable substrate; reduction
of acetylene by nitrogenase is dependent on energy and reducing power. Blockage by
acetylene of the first step of dissimilation of methane prevents the generation of energy
and reducing power. This explanation was further corroborated by the observation that
methanol or other substrates that are oxidizable in the presence of acetylene, support
acetylene reduction by methane-grown cells.
Other pure cultures of methane-oxidizing bacteria also exhibited similar behaviour to
strain 41. Bacteriagrowing on lower hydrocarbons responded similarly to acetylene.Only
the enzyme involved in the first attack on the hydrocarbon was inhibited by acetylene.
Thus the acetylene-ethylene test cannot be employed for measuring nitrogenase activity
46
in methane-utilizing bacteria or inbacteria utilizing lowerhydrocarbons whenalkanesare
the sole energy source.
Alternatives in assaying nitrogenase activity of methane-oxidizing bacteria have been
considered. Acrylonitrile reduction by nitrogenase yields propane, propylene and ammonia as products. However, the predominant hydrocarbon product propylene was cooxidized while acrylonitrile inhibited methane oxidation and growth of the methaneoxidizing bacteria (de Bont, unpublished results).Dalton (personal communication) used
N 2 0 as artificial substrate for nitrogenase. This assay may be of value for pure culture
studies, but in ecological studies the method isnot applicable since the reduction product
isN 2 . Consequently, nitrogenase activity should be measured with 1 S N 2 .
Oxygen sensitivity
In paper I attention is drawn to the oxygen sensitivity of methane-oxidizing bacteria.
Growth of strain 41 in nitrate-containing medium was little influenced by oxygen pressure. But the bacterium was extremely sensitive to oxygen when dependent on N2 as
nitrogen source because of the sensitivity of its nitrogenase towards oxygen. This was
demonstrated by acetylene reduction under different levels of oxygen. A similar behaviour has been observed for other obligate aerobic nitrogen-fixing bacteria. Dalton and
Postgate (1969) reported that Azotobacter chroococcum showed no unusual sensitivity
to oxygen when growing in NH4-containing media. But N2-growing cells were inhibited
by high aeration rates even at atmospheric oxygen pressure. Bacteria of the type of
Mycobacterium flavum are alsosensitive to oxygen when fixing N2 (Bigginsand Postgate,
1969; de Bont and Leijten, 1976). The sensitivity of methane-oxidizing bacteria to
oxygen when fixing N2 was confirmed by Whittenbury, Dalton, Eccleston and Reed
(1975) and Rudd, Furutani, Flett and Hamilton (1976).
Incidence of nitrogenfixation amongmethane-utilizing bacteria
The resultspresented inpaper III show that the capacity to fix atmospheric nitrogen is
common among methane-oxidizing bacteria. Growth appeared readily in enrichments of
nitrogen-fixing methane-oxidizing bacteria that had been set up using inocula from various habitats. Methylosinus-type strains were isolated without difficulty from such enrichments. All these strains resembled strain 41 described in paper I. A number of enrichments also contained Methylomonas-type bacteria. But these organismswere much more
difficult to grow and to isolate than theMethylosinus-type strains.However,the isolation
of these strains was important in demonstrating that both type I and type II methaneoxidizing bacteria may fix nitrogen. Methylomonas-type strain 2possesses the type I
membrane system as opposed to the type II membrane system of strain 41 showing that
nitrogen fixation is not restricted to the type IIorganisms.Pure cultures of Hazeu (1975)
47
including type I as well astype IIrepresentatives were also able to fix nitrogen. Although
Methylosinus and Methylomonas type bacteria appeared inenrichments,inoculated with
soil, mud and ditch water, it was concluded from dilution seriesthat inthe samplesused
as inoculum, coccoid bacteria outnumbered these bacteria. The coccoid bacteria, resembling strain 1 of Hazeu (1975), were very difficult to isolate. They would not grow on
solid agar media. In the enrichments they were overgrown by the fast growingMethylosinusandMethylomonas-type bacteria.
The occurrence in nature of methane-oxidizing bacteria with the capacity to fix nitrogen was investigated by incubating serial dilutions of samples in both nitrogen-free and
nitrate-containing medium. Although nitrate is generally accepted as the best nitrogen
source for methane-oxidizing bacteria, in this experiment it was not found to be a
significantly better source than N 2 . This result indicates that in nature most methaneoxidizing bacteria have the capacity to fix nitrogen.
Ecology of methane-oxidizing bacteria
In nature, methane-oxidizing bacteria are active when methane and oxygen are simultaneously present. Methane is produced in an anaerobic environment by methanogenic
bacteria. The methane thus formed has to diffuse away from the anaerobic zone to an
aerobic environment where it can be oxidized.
Examples of such situations include the aerobic top layer of soil at the mud-water
interface in ditches and ponds that contain oxygenated water. In the underlaying anaerobic mud, methane is produced continuously and may diffuse upwards. In the aerobic
mud-water interfacial region it then may be metabolized by the methane-oxidizing bacteria using oxygen diffusing from the overstanding water. Ratesof methane oxidation in
such systems were estimated employing acetylene as an inhibitor of methane oxidation
(de Bont, unpublished results). Submerged paddy soil waskept in pots and rates of escape
of methane from the pots were measured in the presence and absence of 25ppm acetylene in the atmosphere. There was no increase in the methane content of the atmosphere
above pots without acetylene. Presumably all of the methane diffusing into the aerobic
zone had been oxidized at the soil surface. In the presence of acetylene, this oxidation
was inhibited and methane accumulated in the atmosphere at a rate of about 100nmoles
CH4/cm2 soil surface/day. These observations suggest that oxygen isnot the rate-limiting
factor in such a system. Methane oxidation is limited by the diffusion of methane from
the anaerobic to the aerobic environment. It isnot known whether the methane-oxidizing
bacteria actually fixed nitrogen under these circumstances.
Oxidation of methane may be a very important process in lakes. In the sediments
methane is formed asone of the most important end products of the anaerobic decomposition of organic matter. In stratified lakes, the methane diffuses through the hypolimnion till it reaches the thermocline and epilimnion. Because oxygen isavailable here,the
bacteria can oxidize methane. However, during summer stratification the oxidation of
48
methane was absent from the epilimnion despite adequate methane concentrations.Oxidation was confined to a narrow zone of activity within the thermocline where oxygen
concentrations were low (Rudd, Hamilton and Campbell, 1974; Rudd and Hamilton,
1975). These workers further observed that during autumn and spring overturn, and in
winter, methane oxidation also continued at high oxygen concentrations throughout the
water column. These observations could be explained by considering the oxygen sensitivity of the methane-oxidizing bacteria when dependent on nitrogen fixation. Duringsummer stratification the low concentration of nitrogen compounds limits the activity of the
methane-oxidizing bacteria. Because of the low oxygen concentration in a zone in the
thermocline, nitrogen fixation by the methane-oxidizing bacteria can provide the nitrogen. But in the epilimnion the oxygen tension is too high to allow nitrogen fixation.
During overturn of the lake, ammonia from the hypolimnion becomes available throughout the water column,sothat the methane-oxidizing bacteria can grow without fixing N2
(Rudd, Furutani, Flett and Hamilton, 1976). Attempts to detect nitrogen-fixation in the
thermocline within the zone of methane-oxidizing activity using 15 N 2 were unsuccessful.
The rate of nitrogen fixation wasprobably below the limit of detection with this method
(Rudd, Furutani, Flett and Hamilton, 1976).
The aerobic root-soil interfacial region around the rootsof aquaticplants may form a
third ecological niche where methane oxidation may be an important reaction. Water
plants possess internal gas-filled spaces that form an avenue for diffusion of oxygen from
the phyllosphere to the root system. Partof thisoxygen isused to support respiration of
root tissue and possibly some is lost from the root system and is used in the root-soil
interfacial region for oxidation of reduced substances and microbial respiration. Since
methane is a constituent of flooded soils, the bacteria may use this excess oxygen to
oxidize methane in the root-soil interfacial region. However, in experiments with rice
plants it was found, that the bacterial oxidation of methane in this region was of minor
importance in terms of the amount of methane available, even though there were more
nitrogen-fixing methane-oxidizing bacteria in the rhizosphere soil than inthe surrounding
anaerobic soil(de Bont, unpublished results).
Co-oxidation
Co-oxidation isthe concomitant oxidation of anon-growth substrate during growth of
a microorganism on a utilizable carbon and energy source. It is generally assumed thata
microorganism co-oxidizing a substance is not able to obtain energy from this oxidation
(Horvath, 1972). Oxidation of non-utilizable substrates by resting cell suspension grown
at the expense of substances capable of supporting growth isalsotermed co-oxidation.
The phenomenon of co-oxidation was first reported by Leadbetter and Foster (1958,
1960). They noted that washed cell suspensions of Pseudomonas methanica oxidized
ethane even though the bacterium wasunable to utilize ethane asagrowth substrate.The
products of oxidation of ethane were ethanol, acetaldehyde and acetic acid.Propane and
49
n-butane were not oxidized by washed cells, but they were oxidized when present in
cultures growing on methane. Non-hydrocarbon compounds that ditnot support growth
but caused an increase in oxygen uptake by washed cell suspensions included ethanol and
1-propanol. The observations of Leadbetter and Foster (1960) showed that incorporation
of ethane carbon in cell material by Pseudomonasmethanica was of minor importance.
The major portion of the ethane carbon, after oxidation of 14C ethane,wasrecovered as
volatile C2 compounds.Minor amounts were found ascarbon dioxide or cellmaterial.
Later work by Davey,Whittenbury and Wilkinson (1972) and Patel, Hoare, Hoare and
Taylor (1975)with other type Imethane-oxidizing bacteria indicated that the inability of
these organisms to incorporate substantial amounts of C2-carbon units was due to the
absence of a-ketoglutarate dehydrogenase. The tricarboxylic acid cycle thus cannot be
operative in these bacteria. As a consequence, carbon dioxide was not produced from
acetate and cells incorporated acetate-carbon only in a few amino acids (Patel, Hoare,
Hoare and Taylor, 1975). This situation contrasts with that observed in the type II
methane-oxidizing bacteria. In Methanomonas methanooxidans the tricarboxylic acid
cycle is functional during growth on methane in the presence of acetate. When acetate
was incorporated into the growth medium, the cell yield was greatly enhanced for the
same amount of methane supplied. Utilization of acetate for cellular synthesis was confirmed by showing that both 1-14C and 2-14C acetate were incorporated. 1 4 C0 2 wasalso
released from both radiochemical species of 14C acetate (Wadzinski and Ribbons,
1975b).
Methane hydroxylase catalyses the co-oxidation of hydrocarbon substrates by methane-oxidizing bacteria. Ribbons and Michelover (1970) demonstrated that crude extracts
of Methylococcus capsulatusreadily oxidized ethane inasimilarway to the oxidation of
methane. Both oxygen consumption and NADHoxidation accompanied the oxidation of
ethane by methane hydroxylase. The results in papersI and II indicate that methane
hydroxylase oxidizes ethylene and acetylene aswell.Co-oxidation of these substrates was
adaptive aswasthe oxidation of methane.
Non-hydrocarbon substrates may be oxidized because of the unspecific activity of the
enzymes involved in the metabolism of methanol to carbon dioxide. For instance,methanol dehydrogenase from Methylococcus capsulates (Patel, Bose, Mandy and Hoare,
1972) or methanol oxidase from thisorganism (Wadzinski and Ribbons, 1975a) not only
catalyse the oxidation of methanol. Otherprimary alcoholsare degraded aswellby these
enzymes.
However from suchobservationsit remainsunclear whether the microorganism actually benefits from the oxidation of the co-oxidizable alcohol. The bacterium might utilize
the energy obtained from such an oxidation or it might incorporate the product of the
co-oxidized alcohol into its cell material. From the results given in paper II it can be
concluded that co-oxidation of, for instance, ethanol is beneficial to strain 41. This
conclusion contradicts the general opinion that co-oxidation isofno useto an organism.
Ethanol apparently provides energy and reducing power to the nitrogenase as manifested
by acetylene reduction in the presence of this substrate.Becauseinhibition by acetylene
50
prevents co-oxidation of hydrocarbon compounds by the hydroxylase, it wasnot possible
to determine in thisway whether such co-oxidation yielded energy.
Hydrogenase activity
Paper IV describes a special case of oxidation of a non-growth substrate by methaneoxidizing bacteria. Strain 41 showed hydrogenase activity when the bacterium wasgrown
in a medium containing nitrate,but the enzymic activity increased markedly when bacterial growth was dependent on the fixation of gaseousnitrogen. Apparently, hydrogenase
in this strain is associated with the fixation of nitrogen by the bacterium. Oxidation of
hydrogen gas supported the reduction of acetylene, indicating that its oxidation by
hydrogenase supplies reductant and energy to the nitrogenase. Recently, it was demonstrated that acetylene also inhibited hydrogenase activity by Azotobacter chroococcum
(Smith, Hill and Yates, 1976). Hydrogenase activity of whole cells of this organism was
inhibited for 75% in the presence of 10% acetylene. The influence of acetylene on
hydrogen oxidation by strain 41 wasnot investigated in the present study.
Theacetylene-ethylene assay method
Although the acetylene-ethylene assay is a very sensitive and convenient method for
estimating nitrogenase activity, it does have its drawbacks. Some of these disadvantages
have received attention in the past.
Theoretically the stoichiometric relationship between acetylene reduced and N2 fixed
is 3 : 1. But many investigators observed conversion factors that deviated from this ratio.
The proportions found ranged from 2.3 : 1to 6.9 : 1and evenhigher (Hardy, Burnsand
Holsten, 1973). This stoichiometric relationship should be known for the system under
investigation when absolute valuesfor nitrogen fixation are required.
The diffusion rates of acetylene and ethylene inwater may also seriously influence the
test results, especially in water-saturated soil systems that areassayed under undisturbed
conditions, such as in rice fields. Many reports give estimates on the in situ N input by
nitrogen fixation in rice paddies, but it should be realized that only nitrogen-fixing
activities in the top few centimetres of the field can be recorded asacetylene penetrates
only slowly into the mud. Hindrance of gas diffusion was experimentally demonstrated
by Balandreau, Rinaudo, Ibtissam, Fares-Hamad and Dommergues (1975);Rice and Paul
(1971) considered this theoretically.
Until very recently, the influence of acetylene on the biologicalsystem under investigation had not been considered. The first report providing evidence for the influence of
acetylene on metabolic activities was by Brouzes and Knowles (1971). They demonstrated that acetylene at a partial pressure of 0.05 atmosphere inhibited growth ofClostridium pasteurianum on a medium supplemented with sufficient ammonium nitrogen to
51
completely repress the formation of nitrogenase. Acetylene caused inhibition of cell
proliferation and of cell nitrogen accumulation and prevented the increase in rate of
carbon dioxide evolution normally associated withgrowth.These authors concluded that
it was not clear yet what implications an acetylene inhibition might have for nitrogen
fixation assays. Furthermore they said the results of long-term assays of low-activity
materials should be interpreted with caution until more information is available on the
systems and effects involved. Otherindications that acetylene may interfere with the test
results are available. Elleway, Sabine and Nicholas (1971) studied acetylene reduction by
filtrates of rumen contents. They observed that acetylene inhibited methane production.
But they did not consider the possibility that the test results were influenced by this
inhibition. Raimbault (1975) showed that acetylene at a partial pressure of 0.05 atmosphere stopped all the methane evolution when paddy soil was incubated anaerobically.
Inhibition of methanogenesis in sediments by acetylene was very recently reported by
Oremland and Taylor (1975). These authors discussed the effects of acetylene duringthe
nitrogenase assay. They suspected that these effects cause either an over or an under
estimation of sediment nitrogen fixation rates because methanogenic bacteria probably
play a symbiotic role in nitrogen fixation by anaerobic microbial communities. These
results become even more important in view of the observations of Pine and Barker
(1954). They demonstrated 15 N 2 incorporation by Methanobacterium omelianskii.This
culture was later found to be a symbiotic association of two speciesof bacteria (Bryant,
Wolin, Wolin and Wolfe, 1967), but none of them seems to reduce acetylene (Oremland
and Taylor, 1975). Inhibition of cell metabolism by acetylene may perhaps explain this
contradiction. Another example of interference by acetylene in cell metabolism was
provided by Lloyd Balderston, Sherr and Payne (1976). These authors showed that
reduction of nitrate and nitrite to N2 byPseudomonasperfectomarinus wasinhibited by
acetylene. Reduction of nitrous oxide wasalsoinhibited by acetylene.
Acetylene has also strong effects on plants. It acts as an ethylene analogue and half
maximum effects of acetylene may occur at a concentration of 280 ppm. The plant
hormone ethylene is involved in many biological activitiesof the plant. It playsan important role in many metabolic and physiological systems of the plant. The effects can be
rapid and may occur within 2hours (Abeles, 1973). Consequently, especially when
long-term acetylene-ethylene assays are carried out the test results obtained with intact
plantsmay be influenced.
From the results reported in paper II, together with the information cited from literature, it is obvious that the effects of acetylene on the biological system under study can
no longer be ignored. Acetylene should not be considered as an inert gas except for its
interaction with nitrogenase. Especially when absolute valuesof N2 fixation are required,
the acetylene-ethylene assay should be calibrated with aN-based method.
52
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ADAMSE,A.D., HOEKS,J., BONT,J. A.M.de and KESSEL ,J. M. van (1972). Microbialactivities
insoilnearnaturalgasleaks.Archivfur Mikrobiologie 83,32- 51.
BALANDREAU, J., RINAUDO,G., IBTISSAM FARES-HAMAD and DOMMERGUES,Y. (1975).
Nitrogen fixation in the rhizosphere of rice plants. In Nitrogenfixation by free-livingmicro-organisms,pp.57-70. Edited by W.D.P.Stewart.Cambridge University Press.
BIGGINS,D. R. and POSTGATE,J. R. (1969). Nitrogen fixation by culturesand cell-free extractsof
Mycobacteriumflavum 301.Journalof GeneralMicrobiology 56.181-193.
de BONT, J. A. M.and LEIJTEN, M.W.M.(1976).Nitrogenfixationbyhydrogen-utilizing bacteria.
Archivesof Microbiology107, 235-240.
BROUZES, R. and KNOWLES, R. (1971). Inhibition of growth of Clostridium pasteurianumby
acetylene: implication for nitrogen fixation assay. CanadianJournal of Microbiology 17,
1483-1489.
BRYANT,M.P., WOLIN, E.A., WOLIN,M. J. and WOLFE,R. S. (1967).Methanobacillus omelianskii,asymbiotic association of twospeciesofbacteria.Archivfur Mikrobiologie 59, 20-31.
COTY,V.F. (1967). Atmospheric nitrogen fixation by hydrocarbon-oxidizing bacteria. BiotechnologyandBioengineering 9, 25-32.
DALTON, H. and POSTGATE,J. R. (1969). Effect of oxygen on growth ofAzotobacterchroococcuminbatch and continuous cultures.Journalof GeneralMicrobiology 54, 463—473.
DAVEY, J. F.,WHITTENBURY, R. and WILKINSON,J. F.(1972).Thedistribution inthemethylobacteria of some key enzymes concerned with intermediary metabolism.ArchivfurMikrobiologie
87, 359-366.
DAVIS,J. B.,COTY,V.F. and STANLEY,J. P. (1964). Atmospheric nitrogen fixation by methaneoxidizing bacteria.JournalofBacteriology 88,468-472.
ELLEWAY,R. F., SABINE,J. R. and NICHOLAS,D.J. D. (1971). Acetylene reduction by rumen
microflora.Archivfur Mikrobiologie 76, 277-291.
HARDY,R. W.F., BURNS,R.C. and HOLSTEN,R.D. (1973). Applications of the acetylene-ethyleneassay for measurement of nitrogenfixation.SoilBiologyandBiochemistry5, 47-91.
HARPER, H.J. (1939). The effect of natural gason the growth of microorganisms and the accumulation of nitrogen and organicmatter in the soil.SoilScience48,461-466.
HAZEU,W. (1975). Some cultural and physiological aspects of methane-utilizing bacteria. Antonie
van Leeuwenhoek 41, 121-134.
HOEKS,J. (1972). Effect of leakingnaturalgasonsoilandvegetation inurbanareas. Thesis,Agricultural University,Wageningen.
HORVATH, R. S. (1972). Microbial co-metabolism and the degradation of organic compounds in
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LEADBETTER, E. R. and FOSTER, J.W. (1960). Bacterial oxidation of gaseous alkanes.Archiv fur
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LLOYD BALDERSTON, W., SHERR, B. and PAYNE,W.J. (1976).Blockageby acetylene of nitrous
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504-508.
OREMLAND,R.S. and TAYLOR, B.F.(1975).Inhibition of methanogenesisinmarine sedimentsby
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PATEL,R. N., BOSE,H.R., MANDY,W.J.and HOARE,D.S.(1972). Physiologicalstudies of methane- and methanol-oxidizing bacteria: comparison of a primary alcohol dehydrogenase from
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PINE, M. J. and BARKER, H. A. (1954). Studies on the methane bacteria. XI. Fixation of atmosphericnitrogen byMethanobacterium omelianskii. Journalof Bacteriology 68,589-591.
RAIMBAULT,M. (1975). Etude de l'influence inhibitrice de l'acetylene sur la formation biologique
du methanedansun solderiziere.AnnatesdeMicrobiologic 126A,247-258.
RIBBONS, D. W., and MICHALOVER, J. L. (1970). Methane oxidation by cell-free extracts of
Methylococcuscapsulates. FEBSletters 11,14-44.
RICE,W.A. and PAUL,E.A. (1971). The acetylene reduction assay for measuring nitrogen fixation
inwaterlogged soil.Canadian Journalof Microbiology 17, 1049-1056.
RUDD,J. W.M. and HAMILTON, R.D. (1975). Factors controlling rates of methane oxidation and
the distribution of the methane oxidizers in a small stratified lake.Archivfur Hydrobiologie 75,
522-538.
RUDD,J. W.M., HAMILTON, R.D. and CAMPBELL,N.E.R. (1974). Measurement of microbial
oxidation of methanein lakewater. LimnologyandOceanography 19,519-524.
RUDD,J. W.M., FURUTANI, A., FLETT, R. J. and HAMILTON, R.D. (1976). Factors controling
methane oxidation in shield lakes: The role of nitrogen fixation and oxygen concentration. LimnologyandOceanography 21,357-364.
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261-266.
SMITH,L.A., HILL,S. and YATES,M.G. (1976). Inhibition by acetylene of conventional hydrogenaseinnitrogen-fixing bacteria.Nature 262, 209-210.
WADZINSKI, A.M. and RIBBONS,D.W. (1975a). Oxidation of C, compounds by particulate fractions from Methylococcus capsulatus:properties of methanol oxidase and methanol dehydrogenase.Journalof Bacteriology122,1364-1374.
WADZINSKI,A.M.and RIBBONS,D.W. (1975b). Utilization ofacetatebyMethanomonasmethano
oxidans.Journalof Bacteriology123, 380-381.
WHITTENBURY, R., PHILLIPS,K.C. and WILKINSON,J. F. (1970). Enrichment, isolation and
someproperties ofmethane-utilizing bacteria.Journalof GeneralMicrobiology 61, 205-218.
WHITTENBURY, R., DALTON, H., ECCLESTON,M. and REED,H.L. (1975). The different types
of methane-oxidizing bacteria and some of their more unusual properties. InMicrobialgrowthon
C,-compounds.Proceedings of theInternationalSymposium at Tokyo, Japan, September5, 1974.
p. 1-9. Society of Fermentation Technology,Japan.
54
SUMMARY
Methane occurs abundantly in nature. In the presence of oxygen this gas
may be metabolized by bacteria that are able to use it as carbon and energy
source. Several types of bacteria involved in the oxidation of methane have
been described in literature. Methane-utilizing bacteria have in common that
they can only grow on methane or methanol and not on other carbon compounds. There is much confusion in the literature about the ability of these
bacteria to fix atmospheric nitrogen. The object of the investigations presented in this thesis was to obtain more information about possible nitrogen
fixation by methane-utilizing bacteria.
Paper I is concerned with the isolation and description of the methaneoxidizing bacterium strain 41 of the Methylosinus type. This isolate is a
curved rod, motile in young cultures, but on ageingof the culture motility is
lost and exospores are formed. Only methane or methanol serves as growth
substrate for this bacterium. Attempts to demonstrate nitrogen fixation by
this organism were only successful after it was recognized that (i)the organism was sensitive to oxygen when dependent on N2 as nitrogen source
(ii)nitrogenase activity couldnot be assayedby acetylene reduction when the
bacterium wasgrowingon methane.
Growth of strain 41 in nitrate-containing medium withmethane waslittle
influenced by varying the oxygen pressure. But increasing the oxygen pressure when growing the bacterium in nitrogen-free medium severely reduced
growth. Similar phenomena wereobserved when the bacterium wasgrown on
agar plates. Colony size on nitrate-containing plates was not affected by
varying the oxygen pressure but on nitrogen-free plates the effect of oxygen
became apparant. Incubation in an atmosphere containing 5%oxygen allowed
normal growth on nitrogen-free medium while with 20%oxygen growthwas
only meagre. The nitrogen-fixing colonies that developed sporadically probably were aresult of locallyoccurring clumpsof the inoculated organism.
Evidence of nitrogen fixation by strain 41 when growing with methane
was obtained by using 15 N 2 . Excess IS N percentages of the culture were
measured after the bacterium wasgrown at reduced oxygen pressure innitrogen-free medium with methane and 1S N 2 .
Although strain41 fixed N2 it did not reduce acetylene when growing on
methane. In papers I and II two possible explanations were considered for
this erratic behaviour. It was observed that nitrogenase activity could be
assayed by acetylene reduction whenthe bacterium wasgrowing on methanol
in nitrogen-free medium and that ethylene was co-oxidized by methanegrown cells and not by methanol-grown cells.Inspite of this observation, the
hypothesis that failure of the acetylene-ethylene assay was due to co-oxida-
55
tion of ethylene by the methane-oxidizing bacterium was incorrect because
co-oxidation of ethylene by methane-grown cells was completely prevented
by acetylene.
Acetylene not only completely prevented the co-oxidation of ethylene,
but it alsoinhibited very strongly the oxidation of methane.Thus,the supply
of energy and reducing power to nitrogenase, needed for the reduction of
acetylene, was impeded. This explanation for the failure of the acetyleneethylene assay with methane-grown bacteria was corroborated by the observation that methanol or other substrates oxidizable in the presence of acetylenesupported acetylene reduction bymethane-grown cells.
In paper II, experiments are reported that were undertaken to study the
mechanism of inhibition of methane oxidation by acetylene. Growth of the
bacterium on methanol in a nitrate-containing medium was not affected by
acetylene whereas growth on methane was completely prevented in the presence of acetylene. Apparently, only the first stepinthe degradation route of
methane, the oxidation of methane to methanol, was blocked by acetylene.
This wasalsoshown by the course of the oxygen uptake of whole-cell suspensions of strain 41. Acetylene suppressed methane-dependent oxygen uptake,
whereas it did not influence methanol-dependent oxygen uptake. Interaction
of acetylene with methane hydroxylase, the enzyme involvedin the oxidation
ofmethane to methanol, wasfurther demonstrated by showing that acetylene
also inhibited co-oxidation of methane hydroxylase-dependent co-substrates.
Acetylene itself was only co-oxidized by methane-grown cells. Methanolgrown cells, lacking methane hydroxylase, did not co-oxidize acetylene. Experiments with whole-cell suspensions were undertaken to study the mechanism of inhibition of methane hydroxylase by acetylene. The uptake by such
suspensions of methane and of dissolved oxygen,bothdependent on methane
concentration, was measured. From these experimentsit wastentatively concluded that acetylene inhibited methane oxidation competitively. Other
strains of methane-oxidizing bacteria behaved similarly to strain 41 in that
growth on methane was inhibited by acetylene. Furthermore, bacteria other
than the methane-oxidizing bacteria that could grow on lower hydrocarbons
were inhibited by acetylene aswellwhengrowing onthe alkanebut not when
growing on non-hydrocarbon substrates. Thus the acetylene-ethylene assay
likewise could not be employed for measuring nitrogenase activity in these
bacteria when the alkanewasthe sole energy source.
The study presented in paper III surveyed the nitrogen-fixing capacity
among methane-oxidizing bacteria. Nitrogen-fixing methane-oxidizing bacteria grew readily in enrichment cultures that had been inoculated with
material from various habitats. Methylosinus-type bacteria were most
abundant in such enrichments butMethylomonas-type organismsoccurred as
well. The Methylosinus-type bacteria were isolated without difficulty from
56
the enrichments. These strains all resembled strain 41. They were motile in
young cultures and formed exospores upon ageing,but the cellsizeand shape
varied. Both straight rods and vibrioid forms were identified. The Methylomonas-type bacteria were much more difficult to isolate and to cultivate
than theMethylosinus-type strains.Growth of thesebacteriawasenhanced in
mixed culture with a small motile rod that frequently appeared as a contaminant of pure cultures.Thenitrogen-fixing Methylomonas-type strain 2possessed the type I membrane system as opposed to the type II membrane
system of strain 41. This result shows that nitrogen fixation isnot restricted
to type IImethane-oxidizing bacteria.
The occurrence in nature of methane-oxidizing bacteria withthe capacity
to fix nitrogen was investigated by incubating dilution series of samples in
nitrate-containing and nitrogen-free media. Nitrate was not found to be a
significantly better nitrogen source than N 2 , indicating that the capacity to
fix N2 is common among methane-oxidizing bacteria in nature. The higher
dilutions of the samples contained coccoid bacteria that differed morphologically from the isolated Methylosinus and Methylomonas-type bacteria. Apparently, these coccoid bacteria were more abundant in the mud and soil
samples investigated than were the Methylosinus and Methylomonas-type
bacteria. But in enrichments they were overgrown by these faster growing
organisms. Isolation of the coccoid bacteria was difficult. The first transfer
from the liquid enrichment cultures to plates with nitrogen-free medium
yielded colonies, but subsequent transfers from these colonies onlygrewina
liquid medium. One isolated coccoid organism was found to possess the
type IImembrane system.
Paper IV comprises a study of the hydrogenase activity of strain 41. This
activity was assayed by measuring the uptake of H2 by growing cultures.
There was some hydrogenase activity when the bacterium was growing in
nitrate-containing medium, but the enzymic activity increased markedly
when bacterial growth was dependent on the fixation of N 2 . The function of
the hydrogenase-nitrogenase association was not clear. Hydrogen gas supported the reduction of acetylene, indicating that its oxidation by hydrogenase supplied reductant and energy tothe nitrogenase.
57
SAMENVATTING
Methaan is ruim voorhanden in de natuur. Bij aanwezigheid van zuurstof
kan het omgezet worden door bakterien die in staat zijn het alskoolstof- en
energiebron te gebruiken. In de literatuur zijn verscheidene typen vanbakterien beschreven die bij de oxydatie van methaan betrokken zijn. Methaanoxyderende bakterien hebben gemeen dat ze alleen maar op methaan of
methanol kunnen groeien. Er heerst in de literatuur verwarring omtrent het
vermogen van deze bakterien om stikstof uit de lucht te binden. De onderzoekingen die in dit proefschrift vermeld zijn, hadden tot doel informatie te
verkrijgen betreffende eventuele stikstofbinding door methaanoxyderende
bakterien.
In hoofdstuk I zijn de isolatie en de karakterisering van de methaanoxyderende bakterie stam 41 van het Methylosinus-type beschreven. Dezebakterie is een gebogen staafje dat beweeglijk is in jonge kulturen, maar als de
kultuur ouder wordt gaat de beweeglijkheid verloren en worden exosporen
gevormd. De bakterie groeit alleen maar op methaan of methanol. Stikstofbinding door deze reinkultuur kon pas aangetoond worden nadat het duidelijk geworden was dat (i)de bakterie gevoelig is voor zuurstof alshij stikstof
bindt (ii)de aktiviteit van nitrogenase niet geregistreerd kan worden met
behulp van de acetyleenreduktie-methode wanneer de bakterie op methaan
groeit.
Variatie van de zuurstofspanning had weinig invloed op de groei van
stam 41 in nitraathoudend medium. Maar in stikstofvrij medium werd de
groei sterk geremd door hoge zuurstofspanningen. Dergelijke verschijnselen
deden zich ook voor wanneer debakterie op platen gekweekt werd. De afmeting van de kolonies op platen met nitraat werd niet beinvloed door de
zuurstofspanning. Maar wanneer de bakterie op stikstofvrije platen gekweekt
werd ontstond er normale groei indien de platen onder 5%zuurstof werden
gefnkubeerd, terwijl degroeionder 20%zuurstof zeergeringwas.De stikstofbindende kolonies die hier en daar op zulke platen verschenen waren waarschijnlijk ontstaan als gevolg van plaatselijke ophopingen van de afgestreken
bakterien.
Het vermogen tot stikstofbinding door stam 41bij groeiopmethaan werd
aangetoond met behulp van l s N 2 . Nadat de bakterie op methaan gekweekt
was in stikstofvrij medium onder verlaagde zuurstofspanning konden verhoogde 15N-gehaltenvan de kulturen gemeten worden.
Stam 41 kan geen acetyleen reduceren alshij op methaan groeit hoewel hij
dan wel stikstof kan binden. In de hoofdstukken I en II werden twee mogelijkheden in beschouwing genomen om dit merkwaardige gedrag te kunnen
verklaren. Het falen van de acetyleenreduktiemethode wasniet het gevolgvan
59
de ko-oxydatie van ethyleen. Dit ondanks het feit dat bakterien die in stikstofvrij medium op methanol groeiden wel acetyleen konden reduceren terwijl ze geen ethyleen ko-oxydeerden. Bacterien die op methaan groeiden
ko-oxydeerden weliswaar ethyleen, maar het bleek,dat deze ko-oxydatie volledigverhinderd werd door aanwezigheid van acetyleen.
Niet alleen de ko-oxydatie vanethyleen werd sterk door acetyleen geremd.
Ook de oxydatie van methaan werd er door tot stilstand gebracht. Hierdoor
kon nitrogenase niet langer beschikken overde energieen de reduktie-equivalenten die het nodig heeft om acetyleen te reduceren. Deze verklaring voor
het falen van de acetyleenreduktiemethode bij bakterien die op methaan
groeiden werd nog ondersteund door de waarneming dat deze bakterien wel
acetyleen reduceerden indien ze de beschikking hadden overmethanol of een
ander substraat dat wel in de aanwezigheid van acetyleen geoxydeerd kon
worden.
In hoofdstuk II zijn verder experimenten beschreven die tot doel hadden
het mechanisme van de remming van de methaanoxydatie door acetyleen te
bestuderen. De groei van stam41 in nitraathoudend medium werd nietbei'nvloed door acetyleen indien methanol als substraat was gegeven, maar groei
op methaan werd vollediggeremd door acetyleen.Kennelijk werd de oxydatie
van methaan tot methanol, de eerste stap in de afbraak van methaan tot
koolzuur, onmogelijk gemaakt door acetyleen. Dit kon eveneensworden aangetoond door de zuurstofopname doorgewassen cellenvan stam 41te meten.
Zuurstofopname, veroorzaakt door de oxydatie vanmethaan, werd dooracetyleen verhinderd, maar zuurstofopname gekoppeld aan methanoloxydatie
werd niet door acetyleen geremd. Een interaktie van acetyleen met methaanhydroxylase, het enzymsysteem dat de oxydatie van methaan tot methanol
katalyseert, werd ook nog opandere manieren gedemonstreerd. Het bleek dat
de ko-oxydatie van verbindingen die voor hun oxydatie van methaan-hydroxylase afhankelijk waren eveneens door acetyleen verhinderd werd. Verder
werd nog gevonden dat acetyleen zelf ook geko-oxydeerd werd door cellen
die op methaan gekweekt waren. Maar cellen die op methanol gegroeid waren,
en het enzym methaan-hydroxylase niet bezaten, ko-oxydeerden acetyleen
niet. Het mechanisme van de remming van methaan-hydroxylase door acetyleen werd bestudeerd met behulp van gewassen cellen die opmethaan waren
voorgekweekt. De zuurstofopname en de methaanopname door deze cellen
werden gelijktijdig gemeten. Uit deze experimenten werd onder enigvoorbehoud gekonkludeerd dat acetyleen de methaanoxydatie kompetitief remt.
Andere methaanoxyderende bakterien gedroegen zich op dezelfde manierals
stam 41. Ook hier werd groei op methaan verhinderd door acetyleen.Bakterien die op lagere alkanen groeiden vertoonden een overeenkomstig gedrag.
De groei werd door acetyleen verhinderd,maar dezeverbinding oefende geen
remmende invloed uit op.de ontwikkeling van de bakterien indien andere
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substraten gegeven werden. Als gevolg hiervan kon de acetyleenreduktiemethode voor deze bakterien eveneens niet gebruikt worden indien het alkaan
alsenergiebron dienst deed.
De onderzoekingen vermeld in hoofdstuk HI waren eropgericht het voorkomen van de eigenschap stikstof te kunnen binden bij methaanoxyderende
bakterien nate gaan. Stikstofbindende methaanoxyderende bakterien konden
gemakkelijk worden opgehoopt onder gebruikmaking van entmateriaal van
verschillende herkomst. Bakterien van het Methylosinus-type traden op de
voorgrond in deze ophopingen, maar organismenvanhet Methylomonas-type
kwamen ook voor.Bakterien vanhetMethylosinus-type konden zonder moeite gei'soleerd worden. Zij leken alle op stam 41. In jonge kulturen waren ze
beweeglijk en in oude kulturen werden exosporen gevormd. De vorm en de
afmetingen van de cellen varieerden. Er werden zowel stammen met rechte
staafjes alsmetvibrio-achtige cellen gei'soleerd. Bakterien van het Methylomowas-type waren moeilijker te isoleren en te kweken dan dievanhet Methylosinus-type. De groei van bakterien van de eerstgenoemde groep werd sterk
bevorderd in mengkultuur met een klein beweeglijk staafje dat vaak als
infektie in reinkulturen terecht kwam. De onderzochte stikstofbindende
methaanoxyderende stam 2 van het Methylomonas-typo.had het type Imembraansysteem in tegenstelling tot het type II membraansysteem van stam41.
Hieruit blijkt dat het vermogen om stikstof te binden niet tot de type II
methaanoxyderende bakterien beperkt is.
Het voorkomen in de natuur van methaanoxyderende bakterien die in
staat zijn om stikstof te binden werd onderzocht door verdunningsreeksen
van grond- en watermonsters te inkuberen in zowel nitraathoudend als stikstofvrij medium. Nitraat wasgeenbetere stikstofbron danN 2 , wat er opwees
dat het vermogen tot stikstofbinding eenveelvoorkomende eigenschap is van
methaanoxyderende bakterien. In de hoogste verdunningen van deze monsters kwamen coccen voor die zich duidelijk onderscheidden van degei'soleerde bakterien van deMethylosinus- enMethylomonas-typen. In demodder-en
grondmonsters kwamen deze coccen dus kennelijk in grotere aantallen voor
dan bakterien van de beide andere typen. Maar in de ophopingskulturen
werden de coccen overgroeid door sneller groeiende typen. Isolatie van de
coccen was moeilijk. Op platen met stikstofvrij medium groeiden kolonies na
een eerste overenting uit de vloeibare ophopingskultuur, maar volgende overentingen groeiden slechts in vloeibaar medium. Van een gei'soleerde stamvan
het coccen-type werd vastgesteld dat het type II membraansysteem voorkwam.
Hoofdstuk IV bevat gegevens over hydrogenase-aktiviteit van stam41.
Deze aktiviteit werd bepaald ingroeiende kulturen door de opname van H2 te
meten. Bij groei van de bakterie in nitraathoudend medium was de hydrogenase-aktiviteit gering, maar bij stikstofbindende bakterien was deze aktivi61
teit veel groter. De betekenis van het verband tussenhydrogenase en nitrogenase voor deze bakterie is niet duidelijk. Gevonden werd dat acetyleen gereduceerd kon worden met behulp van waterstof. Hieruit valt af te leiden, dat
nitrogenase van energie en reduktie-equivalenten werd voorzien door de oxydatie van H2 door hydrogenase.
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CURRICULUMVITAE
Op verzoek van het College van Dekanen van de Landbouwhogeschool
volgt hier een korte levensbeschrijving. De auteur van dit proefschrift werd
geboren op 6maart 1947 te Waalwijk. Hij behaalde het H.B.S.-B diploma in
1964. In datzelfde jaar liet hij zich inschrijven alsstudent aan de Landbouwhogeschool. In September 1972 studeerde hij af in derichting waterzuivering
waarna hij wetenschappelijk assistent werd op de afdeling mikrobiologie van
de eerder genoemde hogeschool.
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